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United States Patent |
5,578,572
|
Horwitz
,   et al.
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November 26, 1996
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Anti-gram-positive bacterial methods and materials
Abstract
The present invention relates to methods of treating gram-positive
bacterial infections by administration of a BPI protein product alone, or
in combination with an antibiotic. BPI protein product alone has a
bactericidal or growth inhibitory effect on selected gram-positive
organisms. BPI protein product also increases the susceptibility of
gram-positive organisms to antibiotics and can even reverse resistance of
gram-positive organisms to antibiotic.
Inventors:
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Horwitz; Arnold (Los Angeles, CA);
Lambert, Jr.; Lewis H. (Fremont, CA);
Little, II; Roger G. (Benicia, CA)
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Assignee:
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Xoma Corporation (Berkeley, CA)
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Appl. No.:
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372783 |
Filed:
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January 13, 1995 |
Current U.S. Class: |
514/12; 514/21; 530/350 |
Intern'l Class: |
A61K 038/16; A61K 038/17 |
Field of Search: |
514/12,21
530/350
|
References Cited
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5447914 | Sep., 1995 | Travis et al. | 514/16.
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WO94/20129 | Sep., 1994 | WO.
| |
WO94/20128 | Sep., 1994 | WO.
| |
WO94/20532 | Sep., 1994 | WO.
| |
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|
Primary Examiner: Achutamurthy; Ponnathapura
Attorney, Agent or Firm: Marshall, O'Toole, Gerstein, Murray & Borun
Parent Case Text
This is a continuation-in-part of U.S. patent application Ser. No.
08/274,299 filed Jul. 11, 1994, which is a continuation-in-part of U.S.
patent application Ser. No. 08/209,762 filed Mar. 11, 1994, which is a
continuation-in-part of U.S. patent application Ser. No. 08/183,222 filed
Jan. 14, 1994, abandoned all of which are incorporated herein by reference
.
Claims
What is claimed are:
1. A method of treating a BPI-susceptible gram-positive bacterial infection
comprising the step of administering to a subject suffering from a
BPI-susceptible gram-positive bacterial infection a BPI protein product in
an amount sufficient for monotherapeutic effectiveness.
2. The method of claim 1 wherein the BPI protein product is administered at
a dose of from 1 .mu.g/kg/day to 100 mg/kg/day.
3. The method of claim 2 wherein the BPI protein product is administered at
a dose of from 1 mg/kg/day to 20 mg/kg/day.
4. A method of killing or inhibiting growth of BPI-susceptible
gram-positive bacteria in vitro comprising contacting the bacteria with a
BPI protein product.
5. A method of killing or inhibiting growth of gram-positive bacteria in
vitro comprising contacting the bacteria with a BPI protein product and
one or more antibacterial agents.
6. A method of killing or inhibiting growth of Mycoplasma comprising
contacting the Mycoplasma with a BPI protein product.
7. The method of claim 6 further comprising contacting the Mycoplasma with
antibiotic.
8. In a method of treating a subject suffering from a gram-positive
bacterial infection by administering one or more antibiotics, the
improvement comprising concurrently administering a BPI protein product in
an amount effective to increase antibiotic susceptibility of a
gram-positive bacterial species involved in the infection.
9. The method of claim 8 wherein the BPI protein product is administered at
a dose of from 1 .mu.g/kg/day to 100 mg/kg/day.
10. The method of claim 8 wherein the BPI protein product is administered
at a dose of from 1 mg/kg/day to 20 mg/kg/day.
11. In a method of treating a subject suffering from a gram-positive
bacterial infection by administering one or more antibiotics, the
improvement comprising concurrently administering a BPI protein product in
an amount effective to reverse antibiotic resistance of a gram-positive
bacterial species involved in the infection.
12. The method of claim 11 wherein the BPI protein product is administered
at a dose of from 1 .mu.g/kg/day to 100 mg/kg/day.
13. The method of claim 11 wherein the BPI protein product is administered
at a dose of from 1 mg/kg/day to 20 mg/kg/day.
Description
BACKGROUND OF THE INVENTION
The present invention relates generally to methods and materials for
treating gram-positive bacterial infections by administration of
bactericidal/permeability-increasing (BPI) protein products alone, or in
combination with antibiotic substances. Also disclosed are analogous
methods and materials involving use of lipopolysaccharide binding protein
(LBP) derivatives.
BPI is a protein isolated from the granules of mammalian polymorphonuclear
leukocytes (PMNs or neutrophils), which are blood cells essential in the
defense against invading microorganisms. Human BPI protein has been
isolated from PMNs by acid extraction combined with either ion exchange
chromatography [Elsbach, J. Biol. Chem., 254: 11000 (1979)] or E. coli
affinity chromatography [Weiss, et al., Blood, 69: 652 (1987)]. BPI
obtained in such a manner is referred to herein as natural BPI and has
been shown to have potent bactericidal activity against a broad spectrum
of gram-negative bacteria. The molecular weight of human BPI is
approximately 55,000 daltons (55 kD). The amino acid sequence of the
entire human BPI protein and the nucleic acid sequence of DNA encoding the
protein have been reported in FIG. 1 of Gray et at., J. Biol. Chem., 264:
9505 (1989), incorporated herein by reference. The Gray et al. amino acid
sequence is set out in SEQ ID NO: 69 hereto.
BPI is a strongly cationic protein. The N-terminal half of BPI accounts for
the high net positive charge; the C-terminal half of the molecule has a
net charge of -3. [Elsbach and Weiss (1981), supra.] A proteolytic
N-terminal fragment of BPI having a molecular weight of about 25 kD has an
amphipathic character, containing alternating hydrophobic and hydrophilic
regions. This N-terminal fragment of human BPI possesses the
anti-bacterial efficacy of the naturally-derived 55 kD human BPI
holoprotein. [Ooi et al., J. Bio. Chem., 262: 14891-14894 (1987)]. In
contrast to the N-terminal portion, the C-terminal region of the isolated
human BPI protein displays only slightly detectable anti-bacterial
activity against gram-negative organisms. [Ooi et al., J. Exp. Med., 174:
649 (1991).] An N-terminal BPI fragment of approximately 23 kD, referred
to as "rBPI.sub.23," has been produced by recombinant means and also
retains anti-bacterial activity against gram-negative organisms.
Gazzano-Santoro et al., Infect. Immun. 60: 4754-4761 (1992).
The bactericidal effect of BPI has been reported to be highly specific to
gram-negative species, e.g., in Elsbach and Weiss, Inflammation: Basic
Principles and Clinical Correlates, eds. Gallin et al., Chapter 30, Raven
Press, Ltd. (1992). BPI is commonly thought to be non-toxic for other
microorganisms, including yeast, and for higher eukaryotic cells. Elsbach
and Weiss (1992), supra, reported that BPI exhibits anti-bacterial
activity towards a broad range of gram-negative bacteria at concentrations
as low as 10.sup.-8 to 10.sup.-9 M, but that 100- to 1,000-fold higher
concentrations of BPI were non-toxic to all of the gram-positive bacterial
species, yeasts, and higher eukaryotic cells tested at that time. It was
also reported that BPI at a concentration of 10.sup.-6 M or 160 .mu.g/ml
had no toxic effect, when tested at a pH of either 7.0 or 5.5, on the
gram-positive organisms Staphylococcus aureus (four strains),
Staphylococcus epidermidis, Streptococcus faecalis, Bacillus subtilis,
Micrococcus lysodeikticus, and Listeria monocytogenes. BPI at 10.sup.-6 M
reportedly had no toxic effect on the fungi Candida albicans and Candida
parapsilosis at pH 7.0 or 5.5, and was non-toxic to higher eukaryotic
cells such as human, rabbit and sheep red blood cells and several human
tumor cell lines. See also Elsbach and Weiss, Advances in Inflammation
Research, ed. G. Weissmann, Vol. 2, pages 95-113 Raven Press (1981). This
reported target cell specificity was believed to be the result of the
strong attraction of BPI for lipopolysaccharide (LPS), which is unique to
the outer membrane (or envelope) of gram-negative organisms.
The precise mechanism by which BPI kills gram-negative bacteria is not yet
completely elucidated, but it is believed that BPI must first bind to the
surface of the bacteria through electrostatic and hydrophobic interactions
between the cationic BPI protein and negatively charged sites on LPS. LPS
has been referred to as "endotoxin" because of the potent inflammatory
response that it stimulates, i.e., the release of mediators by host
inflammatory cells which may ultimately result in irreversible endotoxic
shock. BPI binds to lipid A, reported to be the most toxic and most
biologically active component of LPS.
In susceptible gram-negative bacteria, BPI binding is thought to disrupt
LPS structure, leading to activation of bacterial enzymes that degrade
phospholipids and peptidoglycans, altering the permeability of the cell's
outer membrane, and initiating events that ultimately lead to cell death.
[Elsbach and Weiss (1992), supra]. BPI is thought to act in two stages.
The first is a sublethal stage that is characterized by immediate growth
arrest, permeabilization of the outer membrane and selective activation of
bacterial enzymes that hydrolyze phospholipids and peptidoglycans.
Bacteria at this stage can be rescued by growth in serum albumin
supplemented media [Mannion et al., J. Clin. Invest., 85: 853-860 (1990)].
The second stage, defined by growth inhibition that cannot be reversed by
serum albumin, occurs after prolonged exposure of the bacteria to BPI and
is characterized by extensive physiologic and structural changes,
including apparent damage to the inner cytoplasmic membrane.
Initial binding of BPI to LPS leads to organizational changes that probably
result from binding to the anionic groups in the KDO region of LPS, which
normally stabilize the outer membrane through binding of Mg.sup.++ and
Ca.sup.++. Attachment of BPI to the outer membrane of gram-negative
bacteria produces rapid permeabilization of the outer membrane to
hydrophobic agents such as actinomycin D. Binding of BPI and subsequent
gram-negative bacterial killing depends, at least in part, upon the LPS
polysaccharide chain length, with long O-chain bearing, "smooth" organisms
being more resistant to BPI bactericidal effects than short O-chain
bearing, "rough" organisms [Weiss et al., J. Clin. Invest. 65: 619-628
(1980)]. This first stage of BPI action, permeabilization of the
gram-negative outer envelope, is reversible upon dissociation of the BPI,
a process requiring the presence of divalent cations and synthesis of new
LPS [Weiss et al., J. Immunol. 132: 3109-3115 (1984)]. Loss of
gram-negative bacterial viability, however, is not reversed by processes
which restore the envelope integrity, suggesting that the bactericidal
action is mediated by additional lesions induced in the target organism
and which may be situated at the cytoplasmic membrane (Mannion et al., J.
Clin. Invest. 86: 631-641 (1990)). Specific investigation of this
possibility has shown that on a molar basis BPI is at least as inhibitory
of cytoplasmic membrane vesicle function as polymyxin B (In't Veld et al.,
Infection and Immunity 56: 1203-1208 (1988)) but the exact mechanism as
well as the relevance of such vesicles to studies of intact organisms has
not yet been elucidated.
BPI is also capable of neutralizing the endotoxic properties of LPS to
which it binds. Because of its bactericidal properties for gram-negative
organisms and its ability to neutralize LPS, BPI can be utilized for the
treatment of mammals suffering from diseases caused by gram-negative
bacteria, such as bacteremia or sepsis.
U.S. Pat. No. 5,198,541 discloses recombinant genes encoding and methods
for expression of BPI proteins, including BPI holoprotein and fragments of
BPI. It also describes the use of N-terminal fragments of BPI protein for
co-treatment of gram-negative bacterial diseases with certain antibiotics,
specifically penicillin, cephalosporins, rifampicin and actinomycin D.
Antibiotics are natural chemical substances of relatively low molecular
weight produced by various species of microorganisms, such as bacteria
(including Bacillus species), actinomycetes (including Streptomyces) and
fungi, that inhibit growth of or destroy other microorganisms. Substances
of similar structure and mode of action may be synthesized chemically, or
natural compounds may be modified to produce semi-synthetic antibiotics.
These biosynthetic and semi-synthetic derivatives are also effective as
antibiotics. The major classes of antibiotics are (1) the .beta.-lactams,
including the penicillins, cephalosporins and monobactams; (2) the
aminoglycosides, e.g., gentamicin, tobramycin, netilmycin, and amikacin;
(3) the tetracyclines; (4) the sulfonamides and trimethoprim; (5) the
fluoroquinolones, e.g., ciprofloxacin, norfloxacin, and ofloxacin; (6)
vancomycin; (7) the macrolides, which include for example, erythromycin,
azithromycin, and clarithromycin; and (8)other antibiotics, e.g., the
polymycins, chloramphenicol and the lincosamides.
Antibiotics accomplish their anti-bacterial effect through several
mechanisms of action which can be generally grouped as follows: (1) agents
acting on the bacterial cell wall such as bacitracin, the cephalosporins,
cycloserine, fosfomycin, the penicillins, ristocetin, and vancomycin; (2)
agents affecting the cell membrane or exerting a detergent effect, such as
colistin, novobiocin and polymyxins; (3) agents affecting cellular
mechanisms of replication, information transfer, and protein synthesis by
their effects on ribosomes, e.g., the aminoglycosides, the tetracyclines,
chloramphenicol, clindamycin, cycloheximide, fucidin, lincomycin,
puromycin, rifampicin, other streptomycins, and the macrolide antibiotics
such as erythromycin and oleandomycin; (4) agents affecting nucleic acid
metabolism, e.g., the fluoroquinolones, actinomycin, ethambutol,
5-fluorocytosine, griseofulvin, rifamycins; and (5) drugs affecting
intermediary metabolism, such as the sulfonamides, trimethoprim, and the
tuberculostatic agents isoniazid and para-aminosalicylic acid. Some agents
may have more than one primary mechanism of action, especially at high
concentrations. In addition, secondary changes in the structure or
metabolism of the bacterial cell often occur after the primary effect of
the antimicrobial drug.
The penicillins have a characteristic double-ring system composed of a
.beta.-lactam ring, which provides the antibacterial activity, and a
thiazolidene ring. The penicillins are differentiated by a single side
chain that is unique for each penicillin. The compounds are bactericidal
and act by inhibiting bacterial transpeptidase, an enzyme involved in
synthesis of the bacterial cell wall. Because of their mechanism of
action, penicillins are generally active against growing, but not resting,
cells. Penicillins, especially penicillin G, have largely gram-positive
activity; the relative insensitivity of gram-negative rods to penicillin G
and several other penicillins is probably due to the permeability barrier
of the outer membrane of gram-negative bacteria. Ampicillin,
carbenicillin, ticarcillin, and some other penicillins are active against
gram-negative bacteria because they can pass through this outer membrane.
Penicillins have relatively few adverse effects, the most important of
which are the hypersensitivity (allergic) reactions. These compounds are
widely distributed in the body, but do not enter cells and do not usually
accumulate in CSF.
Bacterial resistance to the penicillins is by production of the enzyme
.beta.-lactamase, which catalyzes hydrolysis of the .beta.-lactam ring.
The percentage of bacteria resistant to penicillin has risen to about 80%.
Several penicillins, including methicillin, oxacillin, cloxacillin,
dicloxacillin and nafcillin, are not affected by the .beta.-lactamase of
staphylococci. These antibiotics are useful against most
.beta.-lactamase-producing species of Staphylococcus. However, a small
number of species are resistant even to these penicillins. Some
penicillins, amoxicillin and ticarcillin, are marketed in combination with
clavulanic acid, which is a .beta.-lactamase inhibitor that covalently
binds to the enzyme and prevents it from hydrolyzing the antibiotics.
Another inhibitor, sulbactam, is marketed in combination with ampicillin.
The cephalosporins are characterized by a .beta.-lactam ring, like the
penicillins, but have an adjacent dihydrothiazine ring instead of a
thiazolidene ring. For convenience, these compounds are generally
classified by generations. The first generation includes cephalothin,
cephapirin, cefazolin, cephalexin, cephradine and cefadroxil. These drugs
generally have excellent gram-positive activity except for enterococci and
methicillin-resistant staphylococci, and have only modest gram-negative
coverage. The second generation includes cefamandole, cefoxitin,
ceforanide, cefuroxime, cefuroxime axetil, cefaclor, cefonicid and
cefotetan. This generation generally loses some gram-positive activity by
weight and gains limited gram-negative coverage. The third generation
includes cefotaxime, moxalactam, ceftizoxime, ceftriaxone, cefoperazone
and ceftazidime. These compounds generally sacrifice further gram-positive
activity by weight but gain substantial gram-negative coverage against
Enterobacter and sometimes are active against Pseudoraonas. The
cephalosporins bind to penicillin-binding proteins with varying affinity.
Once binding occurs, protein synthesis is inhibited. Cephalosporins are
usually well tolerated; adverse effects include hypersensitivity reactions
and gastrointestinal effects. Cephalosporins may interact with nephrotoxic
drugs, particularly aminoglycosides, to increase toxicity. Resistance to
cephalosporins is mediated by several mechanisms, including production of
.beta.-lactamase, although some strains that do not produce
.beta.-lactamase are nevertheless resistant.
Imipenem is a N-formimidoyl derivative of the mold product thienamycin. It
contains a .beta.-lactam ring and somewhat resembles penicillin except for
differences in the second ring. It has activity against both gram-positive
and gram-negative organisms and is resistant to most .beta.-lactamases,
although not those from Pseudomonas. It is marketed in combination with
cilastin, a compound that inhibits inactivation of imipenem in the kidney
by renal dihydropeptidase I enzyme. Cilastin increases the concentration
of imipenem in urine, although not in blood.
Aztreonam is the first of a new group of antibiotics referred to as the
monobactams. These agents have a .beta.-lactam ring but lack the second
ring characteristic of the penicillins and cephalosporins. It acts by
binding to penicillin-binding proteins, and produces long, filamentous
bacterial shapes that eventually lyse. Aztreonam is active only against
aerobic gram-negative bacteria, is susceptible to inactivation by some
.beta.-lactamases, and has few adverse effects.
The aminoglycosides contain amino sugars linked to an aminocyclitol ring by
glycosidic bonds. They have similar mechanisms of action and properties,
but differ somewhat in spectrum of action, toxicity, and susceptibility to
bacterial resistance. The compounds are bactericidal, with activity
against both gram-positive and gram-negative organisms, and act by binding
to proteins on the 30S ribosome of bacteria and inhibiting protein
synthesis. The aminoglycosides also bind to isolated LPS and have a very
weak outer membrane permeabilizing effect. [Taber et at., Microbiological
Reviews 53: 439-457 (1987)); Kadurugamuwa et al., Antmicrobial Agents and
Chemotherapy, 37: 715-721 (1993); Vaara, Microbiological Reviews 56:
395-411 (1992)]. This class of antibiotics includes amikacin, gentamicin,
kanamycin, neomycin, netilmycin, paromomycin and tobramycin. The
aminoglycosides are usually reserved for more serious infections because
of severe adverse effects including ototoxicity and nephrotoxicity. There
is a narrow therapeutic window between the concentration required to
produce a therapeutic effect, e.g., 8 .mu.g/ml for gentamicin, and the
concentration that produces a toxic effect, e.g., 12 .mu.g/ml for
gentamicin. Neomycin in particular is highly toxic and is never
administered parenterally.
Tetracyclines have a common four-ring structure and are closely congeneric
derivatives of the polycyclic naphthacenecarboxamide. The compounds are
bacteriostatic, and inhibit protein synthesis by binding to the 30S
subunit of microbial ribosomes and interfering with attachment of
aminoacyl tRNA. The compounds have some activity against both
gram-positive and gram-negative bacteria; however, their use is limited
because many species are now relatively resistant. Adverse effects include
gastrointestinal effects, hepatotoxicity with large doses, and
nephrotoxicity in some patients. This antibiotic class includes
tetracycline, chlortetracycline, demeclocycline, doxycycline,
methacycline, minocycline and oxytetracycline.
The sulfonamides are derivatives of sulfanilamide, a compound similar in
structure to para-aminobenzoic acid (PABA), which is an essential
precursor for bacterial synthesis of folic acid. The compounds are
generally bacteriostatic, and act by competitively inhibiting
incorporation of PABA into tetrahydrofolic acid, which is a required
cofactor in the synthesis of thymidines, purines and DNA. Sulfonamides
have a wide range of activity against gram-positive and gram-negative
bacteria, but their usefulness has diminished with increasingly high
prevalence of bacterial resistance. The sulfonamide class of antibiotics
includes sulfacytine, sulfadiazine, sulfamethizole, sulfisoxazole,
sulfamethoxazole, sulfabenzamide and sulfacetamide. Adverse effects
include hypersensitivity reactions and occasional hematological toxicity.
Trimethoprim is an inhibitor of the dihydrofolate reductase enzyme, which
converts dihydrofolic to tetrahydrofolic acid, a required factor for DNA
synthesis. Adverse effects include gastrointestinal distress and rare
hematological toxicity. Trimethoprim is also available in combination with
sulfamethoxazole (also known as co-trimoxazole). The combination is
usually bactericidal, although each agent singly is usually
bacteriostatic. The combination is the drug of choice for Salmonella
infections, some Shigella infections, E. coli traveler's diarrhea and
Pneumocystis carinii pneumonia.
The fluoroquinolones and quinolones are derivatives of nalidixic acid, a
naphthyridine derivative. These compounds are bactericidal, and impair DNA
replication, transcription and repair by binding to the DNA and
interfering with DNA gyrase, an enzyme which catalyzes negative
supercoiling of DNA. The fluoroquinolones, which include norfloxacin,
ciprofloxacin, and ofloxacin, and the quinolones, which include cinoxacin,
have a broad spectrum of antimicrobial activity against gram-negative and
gram-positive organisms. These compounds distribute widely through
extravascular tissue sites, have a long serum half-life, and present few
adverse effects. Because of their effect on DNA, the drugs are
contraindicated in pregnant patients and in children whose skeletal growth
is incomplete.
Vancomycin is a glycopeptide, with a molecular weight of about 1500,
produced by a fungus. It is primarily active against gram-positive
bacteria. The drug inhibits one of the final steps in synthesis of the
bacterial cell wall, and is thus effective only against growing organisms.
It is used to treat serious infections due to gram-positive cocci when
penicillin G is not useful because of bacterial resistance or patient
allergies. Vancomycin has two major adverse effects, ototoxicity and
nephrotoxicity. These toxicities can be potentiated by concurrent
administration of another drug with the same adverse effect, such as an
aminoglycoside.
The macrolides are bacteriostatic and act by binding to the 50S subunit of
70S ribosomes, resulting in inhibition of protein synthesis. They have a
broad spectrum of activity against gram-positive and gram-negative
bacteria and may be bacteriostatic or bactericidal, depending on the
concentration achieved at sites of infection. The compounds distribute
widely in body fluids. Adverse effects include gastrointestinal distress
and rare hypersensitivity reactions. The most common macrolide used is
erythromycin, but the class includes other compounds such as
clarithromycin and azithromycin.
The polymyxins are a group of closely related antibiotic substances
produced by strains of Bacillus polymyxa. These drugs, which are cationic
detergents, are relatively simple, basic peptides with molecular weights
of about 1000. Their antimicrobial activity is restricted to gram-negative
bacteria. They interact strongly with phospholipids and act by penetrating
into and disrupting the structure of cell membranes. Polymyxin B also
binds to the lipid A portion of endotoxin and neutralizes the toxic
effects of this molecule. Polymyxin B has severe adverse effects,
including nephrotoxicity and neurotoxicity, and should not be administered
concurrently with other nephrotoxic or neurotoxic drugs. The drug thus has
limited use as a therapeutic agent because of high systemic toxicity, but
may be used for severe infections, such as Pseudomonas aeruginosa
meningitis, that respond poorly to other antibiotics.
Chioramphenicol inhibits protein synthesis by binding to the 50S ribosomal
subunit and preventing binding of aminoacyl tRNA. It has a fairly wide
spectrum of antimicrobial activity, but is only reserved for serious
infections, such as meningitis, typhus, typhoid fever, and Rocky Mountain
spotted fever, because of its severe and fatal adverse hematological
effects. It is primarily bacteriostatic, although it may be bactericidal
to certain species.
Lincomycin and clindamycin are lincosamide antimicrobials. They consist of
an amino acid linked to an amino sugar. Both inhibit protein synthesis by
binding to the 50S ribosomal subunit. They compete with erythromycin and
chloramphenicol for the same binding site but in an overlapping fashion.
They may be bacteriostatic or bactericidal, depending on relative
concentration and susceptibility. Gastrointestinal distress is the most
common side effect. Other adverse reactions include cutaneous
hypersensitivity, transient hematological abnormalities, and minor
elevations of hepatic enzymes. Clindamycin is often the drug of choice for
infections caused by anaerobic bacteria or mixed aerobic/anaerobic
infections, and can also be used for susceptible aerobic gram-positive
cocci.
Some drugs, e.g. aminoglycosides, have a small therapeutic window. For
example, 2 to 4 .mu.g/ml of gentamicin or tobramycin may be required for
inhibition of bacterial growth, but peak concentrations in plasma above 6
to 10 .mu.g/ml may result in ototoxicity or nephrotoxicity. These agents
are more difficult to administer because the ratio of toxic to therapeutic
concentrations is very low. Antimicrobial agents that have toxic effects
on the kidneys and that are also eliminated primarily by the kidneys, such
as the aminoglycosides or vancomycin, require particular caution because
reduced elimination can lead to increased plasma concentrations, which in
turn may cause increased toxicity. Doses of antimicrobial agents that are
eliminated by the kidneys must be reduced in patients with impaired renal
function. Similarly, dosages of drugs that are metabolized or excreted by
the liver, such as erythromycin, chloramphenicol, or clindamycin, must be
reduced in patients with decreased hepatic function.
Antibiotic resistance in bacteria is an increasingly troublesome problem.
The accelerating development of antibiotic-resistant bacteria, intensified
by the widespread use of antibiotics in farm animals and overprescription
of antibiotics by physicians, has been accompanied by declining research
into new antibiotics with different modes of action. [Science, 264:
360-374 (1994).]Antibiotic resistance, once acquired, can be rapidly
spread to other bacteria, including bacteria of a different species. There
are some species of bacteria that are resistant to all but one antibiotic;
it may be only a matter of time before the appearance of bacterial strains
that are resistant to all antibiotics.
Bacteria acquire resistance to antibiotics through several mechanisms: (1)
production of enzymes that destroy or inactivate the antibiotic [Davies,
Science, 264: 375-381 (1994)]; (2) synthesis of new or altered target
sites on or within the cell that are not recognized by the antibiotic
[Spratt, Science, 264: 388-393 (1994)]; (3) low permeability to
antibiotics, which can be reduced even further by altering cell wall
proteins, thus restricting access of antibiotics to the bacterial
cytoplasmic machinery; (4) reduced intracellular transport of the drug;
and (5) increased removal of antibiotics from the cell via
membrane-associated pumps [Nikaido, Science, 264: 382-387 (1994)].
The susceptibility of a bacterial species to an antibiotic is generally
determined by two microbiological methods. A rapid but crude procedure
uses commercially available filter paper disks that have been impregnated
with a specific quantity of the antibiotic drug. These disks are placed on
the surface of agar plates that have been streaked with a culture of the
organism being tested, and the plates are observed for zones of growth
inhibition. A more accurate technique, the broth dilution susceptibility
test, involves preparing test tubes containing serial dilutions of the
drug in liquid culture media, then inoculating the organism being tested
into the tubes. The lowest concentration of drug that inhibits growth of
the bacteria after a suitable period of incubation is reported as the
minimum inhibitory concentration.
The resistance or susceptibility of an organism to an antibiotic is
determined on the basis of clinical outcome, i.e., whether administration
of that antibiotic to a subject infected by that organism will
successfully cure the subject. While an organism may literally be
susceptible to a high concentration of an antibiotic in vitro, the
organism may in fact be resistant to that antibiotic at physiologically
realistic concentrations. If the concentration of drug required to inhibit
growth of or kill the organism is greater than the concentration that can
safely be achieved without toxicity to the subject, the microorganism is
considered to be resistant to the antibiotic. To facilitate the
identification of antibiotic resistance or susceptibility using in vitro
test results, the National Committee for Clinical Laboratory Standards
(NCCLS) has formulated standards for antibiotic susceptibility that
correlate clinical outcome to in vitro determinations of the minimum
inhibitory concentration of antibiotic.
There continues to exist a need in the art for new antimicrobial, and
especially anti-gram-positive microbial, methods and materials. Products
and methods responsive to this need would ideally involve substantially
non-toxic compounds available in large quantities by means of synthetic or
recombinant methods. Ideal compounds would have bactericidal or
bacteriostatic activity when administered or applied as the sole
anti-microbial agent. Such compounds ideally would also be useful in
combinative therapies with other anti-microbial agents, particularly where
potentiating effects are provided.
SUMMARY OF THE INVENTION
The present invention provides methods and compositions for treating
gram-positive bacterial infections, including conditions associated
therewith or resulting therefrom (for example, sepsis or bacteremia), in a
subject by administering a BPI protein product alone, or concurrently with
an antibiotic. The invention is based upon the discovery that BPI protein
products surprisingly have direct bactericidal and growth inhibitory
effects on some gram-positive organisms, and that BPI protein products
unexpectedly have the ability to increase the antibiotic susceptibility of
gram-positive bacteria, including the ability to reverse in many instances
the antibiotic resistance of gram-positive bacteria. The invention is also
based upon the finding that BPI protein products and antibiotics provide
additive and synergistic bactericidal/growth inhibitory effects when
administered concurrently.
According to one aspect of the invention, a method is provided of treating
a gram-positive bacterial infection comprising the step of administering
to a subject suffering from a gram-positive bacterial infection a BPI
protein product in an amount sufficient for monotherapeutic effectiveness.
This method may be practiced when any BPI-susceptible gram-positive
bacterial species is involved in the infection.
A second aspect of the invention provides a method of treating
gram-positive bacterial infection by concurrently administering to a
subject suffering from a gram-positive bacterial infection a BPI protein
product in an amount sufficient for combinative therapeutic effectiveness
and one or more antibiotics in amounts sufficient for combinative
therapeutic effectiveness. This method is effective even for gram-positive
organisms that are not susceptible to the direct bactericidal/growth
inhibitory effects of BPI.
For concurrent administration with antibiotics, the BPI protein product may
be administered in an amount effective to increase the antibiotic
susceptibility of a gram-positive bacterial species involved in the
infection, or to potentiate the effects of the antibiotic. The BPI protein
product may also be administered in an amount effective to reverse the
antibiotic resistance of a gram-positive bacterial species involved in the
infection. The BPI protein product and the antibiotics may each be
administered in amounts that would be sufficient for monotherapeutic
effectiveness upon administration alone or may be administered in less
than monotherapeutic amounts.
Another aspect of the invention provides a method of treating gram-positive
bacterial infection by concurrently administering to a subject suffering
from a gram-positive bacterial infection a BPI protein product and one or
more antibiotics, in synergistically effective amounts.
In addition, the invention provides a method of killing or inhibiting
growth of gram-positive bacteria comprising contacting the bacteria with a
BPI protein product alone, or in combination with another antibacterial
agent. This method can be practiced in vivo or in a variety of in vitro
uses such as use in food preparation, to decontaminate fluids and surfaces
or to sterilize surgical and other medical equipment and implantable
devices, including prosthetic joints. These methods can correspondingly be
used for in situ sterilization of indwelling invasive devices such as
intravenous lines and catheters, which are often foci of infection, or for
sterilization of in vitro tissue culture media.
The invention further provides a method of killing or inhibiting growth of
gram-positive bacteria with disrupted cell walls, including L-phase
variants, by contacting such bacteria with a BPI protein product alone, or
in conjunction with another antibacterial agent. This method can also be
practiced on organisms that lack cell walls, such as Mycoplasma or
Ureaplasma.
The invention further provides the use of BPI protein product for the
manufacture of a medicament for treatment of gram-positive bacterial
infections, and the use of a BPI protein product in combination with an
antibiotic for manufacture of a medicament for treatment of gram-positive
bacterial infections.
Numerous additional aspects and advantages of the invention will become
apparent to those skilled in the art upon consideration of the following
detailed description of the invention which describes presently preferred
embodiments thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 relates to plate assays of the bactericidal effect of rBPI.sub.23 on
Bacillus subtilis.
FIG. 2 shows results from radial diffusion assays of the growth inhibitory
effect of rBPI.sub.21 on Staphylococcus aureus with or without penicillin.
FIG. 3 depicts results from radial diffusion assays of the effect of
rBPI.sub.21 on penicillin-treated S. aureus and L-phase variants.
FIG. 4 shows the effect of various BPI protein products on
penicillin-treated S. aureus in a radial diffusion assay.
FIGS. 5, 6, 7 and 8 show the effect of various BPI protein products,
including BPI-derived peptides, on the S. aureus bacterial form and
L-phase variant in a radial diffusion assay.
FIG. 9 relates to the effect of CaCl.sub.2 concentration on the growth of
S. aureus L-phase variant in broth.
FIG. 10 depicts results from a broth growth inhibition assay of the effect
of rBPI.sub.21 on S. aureus L-phase variant.
FIGS. 11, 12 and 13 shows results from broth growth inhibition assays of
the effect of rBPI.sub.21 on S. aureus L-phase variant, at CaCl.sub.2
concentrations of 2.5 mM, 5 mM and 10 mM, respectively.
FIG. 14 shows the effect of rBPI.sub.21 in an E. coli broth growth
inhibition assay.
FIG. 15 displays the effect of a variety of BPI protein products on S.
pneumoniae L-forms in a radial diffusion assay.
FIGS. 16 and 17 show the effect of BPI protein products, including
BPI-derived peptides, on the S. pneumoniae bacterial form and L-phase
variant in a radial diffusion assay.
FIGS. 18 and 19 show the effect of BPI protein products, including
BPI-derived peptides, on the S. pyogenes bacterial form and L-phase
variant in a radial diffusion assay.
FIG. 20 shows the effect of a variety of BPI protein products on E.
faecalis L-forms in a radial diffusion assay.
FIGS. 21 and 22 show the effect of BPI protein products, including
BPI-derived peptides, on the mycoplasma Acholeplasma laidlawii in a radial
diffusion assay.
FIGS. 23, 24 and 25 display the effect of BPI protein products and LBP
derivatives on S. aureus L-forms and S. pneumoniae L-forms in radial
diffusion assays.
FIGS. 26 through 32 displays rBPI.sub.21, potentiation of the early
bactericidal effect of various antibiotics on S. pneumoniae, S. aureus and
E. faecalis.
DETAILED DESCRIPTION
The present invention relates to methods and materials for treating
subjects suffering from gram-positive bacterial infections. "Gram-positive
bacterial infection," as used herein, encompasses conditions associated
with or resulting from gram-positive bacterial infection (e.g., sequelae).
These conditions include gram-positive sepsis and one or more of the
conditions associated therewith, including bacteremia, fever, hypotension,
shock, metabolic acidosis, disseminated intravascular coagulation and
related clotting disorders, anemia, thrombocytopenia, leukopenia, adult
respiratory distress syndrome and related pulmonary disorders, renal
failure and related renal disorders, hepatobiliary disease and central
nervous system disorders. These conditions also include translocation of
gram-negative bacteria from the intestines and concomitant release of
endotoxin. Gram-positive bacteria include bacteria from the following
species: Staphylococcus, Streptococcus, Micrococcus, Peptococcus,
Peptostreptococcus, Enterococcus, Bacillus, Clostridium, LactobacilIus,
Listeria, Erysipelothrix, Propionibacterium, Eubacterium, and
Corynebacterium.
A variety of gram-positive organisms are capable of causing sepsis. The
most common organisms involved in sepsis are Staphylococcus aureus,
Streptoccocus pneumoniae, coagulase-negative staphylococci, beta-hemolytic
streptococci, and enterococci, but any gram-positive organism may be
involved. [Bone, J. Critical Care, 8: 51-59 (1993).]
According to one aspect of the invention, BPI protein product alone, in an
amount sufficient for monotherapeutic effectiveness, may be administered
to a subject suffering from infection involving a BPI-susceptible
gram-positive bacteria. When used to describe administration of BPI
protein product alone, the term "amount sufficient for monotherapeutic
effectiveness" means an amount of BPI protein product that provides
bactericidal or growth inhibitory effects when administered as a
monotherapy. The invention utilizes any of the large variety of BPI
protein products known to the art including natural BPI protein,
recombinant BPI protein, BPI fragments, BPI analogs, BPI variants, and
BPI-derived peptides.
This aspect of the invention is based on the discovery that BPI protein
products have direct bactericidal or growth inhibitory activity against
some gram-positive organisms. BPI protein products are also shown herein
to have direct bactericidal or growth inhibitory effects on L-phase
variants of a variety of gram-positive organisms; these L-phase variants
lack the cell walls of the normal bacterial form. BPI protein products are
also expected to exert direct bactericidal/growth inhibitory effects on
the cell wall-less Mycoplasma and Ureaplasma, organisms involved
clinically in respiratory and urogenital infections. Mycoplasma is also a
major contaminant of in vitro tissue cultures.
According to a second aspect of the invention, a subject suffering from a
gram-positive bacterial infection may be treated by concurrent
administration of a BPI protein product in an amount sufficient for
combinative therapeutic effectiveness and one or more antibiotics in
amounts sufficient for combinative therapeutic effectiveness. This aspect
of the invention contemplates concurrent administration of BPI protein
product with any antibiotic or combinations of antibiotics, including
.beta.-lactam antibiotics with and without .beta.-lactamase inhibitors,
aminoglycosides, tetracyclines, sulfonamides and trimethoprim, vancomycin,
macrolides, fluoroquinolones and quinolones, polymyxins and other
antibiotics.
This aspect of the invention is based on the discovery that administration
of BPI protein products improves the therapeutic effectiveness of
antibiotics, e.g., by increasing the antibiotic susceptibility of
gram-positive organisms to a reduced dosage of antibiotics providing
benefits in reduction of cost of antibiotic therapy and/or reduction of
risk of toxic responses to antibiotics. BPI protein products are shown
herein to lower the minimum concentration of antibiotics needed to inhibit
in vitro growth of gram-positive organisms at 24 hours. In cases where BPI
protein product did not affect growth at 24 hours, BPI protein product was
shown to potentiate the early bactericidal effect of antibiotics in vitro
at 0-7 hours. The BPI protein products exert these effects even on
gram-positive organisms that are not susceptible to the direct
bactericidal or growth inhibitory effects of BPI protein product alone.
This aspect of the invention is correlated to the additional discovery that
administration of a BPI protein product can effectively reverse the
antibiotic resistance of a gram-positive organism. BPI protein products
are shown herein to reduce the minimum inhibitory concentration of
antibiotics from a level within the clinically resistant range to a level
within the clinically susceptible range. BPI protein products thus can
convert normally antibiotic-resistant organisms into
antibiotic-susceptible organisms.
According to this second aspect of the invention, the BPI protein product
and antibiotics are concurrently administered in amounts sufficient for
combinative therapeutic effectiveness. When used to describe
administration of a BPI protein product in conjunction with an antibiotic,
the term "amount sufficient for combinative therapeutic effectiveness"
with respect to the BPI protein product means at least an amount effective
to increase the susceptibility of the organism to the antibiotic, and the
term "amount sufficient for combinative therapeutic effectiveness" with
respect to an antibiotic means at least an amount of the antibiotic that
produces bactericidal or growth inhibitory effects when administered in
conjunction with that amount of BPI protein product. Either the BPI
protein product or the antibiotic, or both, may be administered in an
amount below the level required for monotherapeutic effectiveness against
a gram-positive bacterial infection. The BPI protein product may be
administered in an amount which is not sufficient for monotherapeutic
effectiveness but which provides increased antibiotic susceptibility or
potentiates the effects of the antibiotic, or which reverses the
resistance of the gram-positive organism to an antibiotic.
A further aspect of the invention relates to the discovery that concurrent
administration of BPI protein products with antibiotics provides
synergistic bactericidal or growth inhibitory effects beyond the
individual bactericidal or growth inhibitory effects of the BPI protein
product or the antibiotic. Some methods for evaluating the effect of
antimicrobial combinations are described in Eliopoulos and Moellering In
Antibiotics in Laboratory Medicine, 3rd ed. (Lorian, V., Ed.) pp. 432-492,
Williams and Wilkins, Baltimore Md. (1991). There is general agreement on
qualitative definition of synergism: the combined effect of the drugs
being examined is significantly greater than the expected result based on
independent effects of the drugs when used separately. In a checkerboard
assay, the combination of BPI protein product with antibiotics may be
shown to result in a "synergistic" fractional inhibitory concentration
index (FIC). The checkerboard method is based on additivity, which assumes
that the result observed with multiple drugs is the sum of the separate
effects of the drugs being tested; according to this system a FIC of less
than 0.5 is scored as synergy, 1 is scored as additive, and greater than 1
but less than 2 is scored as indifferent. In contrast, kinetic assays are
based on the idea that only one metabolic pathway at a time can be growth
rate-limiting for an organism; according to this system, the combined
effect of drugs that do not interact with one another (autonomous or
indifferent) is simply the effect of the most active drug alone.
According to this aspect of the invention, "effective synergy" or
potentiation upon concurrent administration of BPI protein product with
one or more antibiotics can be obtained in a number of ways. A BPI protein
product may convert an antibiotic-resistant organism into an
antibiotic-susceptible organism or otherwise improve the antibiotic
susceptibility of that organism. Conversely, a BPI-potentiating
antibiotic, such as an antibiotic that acts on the cell wall or cell
membrane of bacteria, may convert a BPI-resistant organism into a
BPI-susceptible organism. Alternatively, the BPI protein product and
antibiotic may both co-potentiate the other agent's activity. The BPI
protein product and antibiotic may have a therapeutic effect when both are
given in doses below the amounts sufficient for monotherapeutic
effectiveness.
Either the BPI protein product or the antibiotics may be administered
systemically or topically. Systemic routes of administration include oral,
intravenous, intramuscular or subcutaneous injection (including into a
depot for long-term release), intraocular or retrobulbar, intrathecal,
intraperitoneal (e.g. by intraperitoneal lavage), transpulmonary using
aerosolized or nebulized drug, or transdermal. Topical routes include
administration in the form of salves, ophthalmic drops, ear drops, or
irrigation fluids (for, e.g., irrigation of wounds).
"Concurrent administration," or co-treatment, as used herein includes
administration of the agents, in conjunction or combination, together, or
before or after each other. The BPI protein product and antibiotics may be
administered by different routes. For example, the BPI protein product may
be administered intravenously while the antibiotics are administered
intramuscularly, intravenously, subcutaneously, orally or
intraperitoneally. Alternatively, the BPI protein product may be
administered intraperitoneally while the antibiotics are administered
intraperitoneally or intravenously, or the BPI protein product may be
administered in an aerosolized or nebulized form while the antibiotics are
administered, e.g., intravenously. The BPI protein product and antibiotics
are preferably both administered intravenously. The BPI protein product
and antibiotics may be given sequentially in the same intravenous line,
after an intermediate flush, or may be given in different intravenous
lines. The BPI protein product and antibiotics may be administered
simultaneously or sequentially, as long as they are given in a manner
sufficient to allow both agents to achieve effective concentrations at the
site of infection.
Concurrent administration of BPI protein product and antibiotic is expected
to provide more effective treatment of gram-positive bacterial infection.
Concurrent administration of the two agents may provide greater
therapeutic effects in vivo than either agent provides when administered
singly. It may permit a reduction in the dosage of one or both agents with
achievement of a similar therapeutic effect. Alternatively, the concurrent
administration may produce a more rapid or complete
bactericidal/bacteriostatic effect than could be achieved with either
agent alone.
Therapeutic effectiveness is based on a successful clinical outcome, and
does not require that the antimicrobial agent or agents kill 100% of the
organisms involved in the infection. Success depends on achieving a level
of antibacterial activity at the site of infection that is sufficient to
inhibit the bacteria in a manner that tips the balance in favor of the
host. When host defenses are maximally effective, the antibacterial effect
required may be minimal. Reducing organism load by even one log (a factor
of 10) may permit the host's own defenses to control the infection. In
addition, augmenting an early bactericidal/bacteriostatic effect can be
more important than long-term bactericidal/bacteriostatic effect. These
early events are a significant and critical part of therapeutic success,
because they allow time for host defense mechanisms to activate.
Increasing the bactericidal rate may be particularly important for
infections such as meningitis, bone or joint infections. [Stratton,
Antibiotics in Laboratory Medicine, 3rd ed. (Loftan, V., Ed.) pp. 849-879,
Williams and Wilkins, Baltimore Md. (1991)].
The effect of BPI protein product to improve the therapeutic effectiveness
of antibiotics in vivo may be demonstrated in in vivo animal models, or
may be predicted on the basis of a variety of in vitro tests, including
(1) determinations of the minimum inhibitory concentration (MIC) of an
antibiotic required to inhibit growth of a gram-negative organism for 24
hours, (2) determinations of the effect of an antibiotic on the kinetic
growth curve of a gram-negative organism, and (3) checkerboard assays of
the MIC of serial dilutions of antibiotic alone or in combination with
serial dilutions of BPI protein product. Exemplary models or tests are
described in Eliopoulos and Moellering In Antibiotics in Laboratory
Medicine, 3rd ed. (Loftan, V., Ed.) pp. 432-492, Williams and Wilkins,
Baltimore Md. (1991).
Using in vitro determinations of antibiotic MIC at 24 hours, a BPI protein
product may be shown to reduce the MIC of the antibiotic. With this
result, it is expected that concurrent administration of the BPI protein
product in vivo will increase susceptibility of the gram-negative organism
to the antibiotic. A BPI protein product may also be shown to reduce the
MIC of an antibiotic from the range in which the organism is considered
clinically resistant to a range in which the organism is considered
clinically susceptible. With this result, it is expected that concurrent
administration in vivo of the BPI protein product with the antibiotic will
reverse resistance and effectively convert the antibiotic-resistant
organism into an antibiotic-susceptible organism.
By measuring the effect of antibiotics on the in vitro growth curves of
gram-negative organisms, in the presence or absence of a BPI protein
product, the BPI protein product may be shown to enhance the early
antibacterial effect of antibiotics at 0-24 hours. Enhancement of early
bactericidal/growth inhibitory effects is important in determining
therapeutic outcome.
BPI protein product is thought to interact with a variety of host defense
elements present in whole blood or serum, including complement, p15 and
LBP, and other cells and components of the immune system. Such
interactions may result in potentiation of the activities of BPI protein
product. Because of these interactions, BPI protein products can be
expected to exert even greater activity in vivo than in vitro. Thus, while
in vitro tests are predictive of in vivo utility, absence of activity in
vitro does not necessarily indicate absence of activity in vivo. For
example, BPI has been observed to display a greater bactericidal effect on
gram-negative bacteria in whole blood or plasma assays than in assays
using conventional media. [Weiss et al., J. Clin. Invest. 90: 1122-1130
(1992)]. This may be because conventional in vitro systems lack the blood
elements that facilitate or potentiate BPI's function in vivo, or because
conventional media contain higher than physiological concentrations of
Mueller-Hinton has.about.physiological conclusion as serum magnesium and
calcium, which are typically inhibitors of the antibacterial activity of
BPI protein products. Furthermore, in the host, BPI protein product is
available to neutralize translocation of gram-negative bacteria and
concomitant release of endotoxin, a further clinical benefit not seen in
or predicted by in vitro tests.
It is also contemplated that the BPI protein product be administered with
other products that potentiate the bactericidal activity of BPI protein
products. For example, serum complement potentiates the gram-negative
bactericidal activity of BPI protein products; the combination of BPI
protein product and serum complement provides synergistic
bactericidal/growth inhibitory effects. See, e.g., Ooi et al. J. Biol.
Chem., 265: 15956 (1990) and Levy et al. J. Biol. Chem., 268: 6038-6083
(1993) which address naturally-occurring 15 kD proteins potentiating BPI
antibacterial activity. See also co-owned, co-pending PCT Application No.
US 94/07834 fled Jul. 13, 1994, which corresponds to U.S. patent
application Ser. No. 08/274,303 filed Jul. 11, 1994 as a
continuation-in-part of U.S. patent application Ser. No. 08/093,201 filed
Jul. 14, 1993. These applications, which are all incorporated herein by
reference, describe methods for potentiating gram-negative bactericidal
activity of BPI protein products by administering lipopolysaccharide
binding protein (LBP) and LBP protein products. LBP protein derivatives
and derivative hybrids which lack CD-14 immunostimulatory properties are
described in PCT Application No. US94/06931 filed Jun. 17, 1994, which
corresponds to co-owned, co-pending U.S. patent application Ser. No.
08/261,660, filed Jun. 17, 1994 as a continuation-in-part of U.S. patent
application Ser. No. 08/079,510, filed Jun. 17, 1993, the disclosures of
all of which are incorporated herein by reference.
BPI is commonly thought to be non-cytotoxic for many types of gram-positive
bacteria. This lack of toxicity may be due primarily to the low affinity
of BPI for the gram-positive cell wall and the ability of the cell wall to
protect the cytoplasmic membrane from prolonged exposure to and subsequent
damage by BPI. Because prokaryotic cytoplasmic membranes from both
gram-positive and gram-negative bacteria may have similar structures that
are distinct from those of eukaryotic membranes, BPI protein products may
also be cytotoxic for gram-positive bacteria if the cell wall is removed
or damaged or if the BPI protein products can be targeted to the cell
surface with high affinity.
Without being bound by a theory of the invention, it is believed that BPI
protein product may have several mechanisms of action. BPI protein product
may act directly on the cell walls of gram-positive bacteria by binding to
LPS-like molecules such as cell wall peptidoglycans and teichoic acid. If
BPI is allowed to reach the inner cytoplasmic membrane, the amphipathic
nature of BPI may allow it to penetrate the cytoplasmic membrane and exert
a bactericidal effect. Thus, agents that act on or disrupt the cell walls
of bacteria such as antibiotics, detergents or surfactants,
anti-peptidoglycan antibodies, anti-lipoteichoic acid antibodies and
lysozyme, may potentiate the activity of BPI by allowing access to the
inner cytoplasmic membrane.
Likewise, BPI's action on the inner cytoplasmic membrane of bacteria may
potentiate the action of antibiotics by allowing penetration of the
antibiotic through the inner membrane, thus permitting it to affect the
bacterial biochemical machinery. Moreover, because gram-positive bacterial
infection may cause stress-induced translocation of bowel flora and/or
LPS, BPI may also act beneficially by killing the gram-negative bacteria
and neutralizing the LPS.
BPI protein product, through its heparin-binding ability, may interfere
with the binding of gram-positive bacteria to the extracellular matrix
(ECM) and to host cells. It has been previously shown that a number of
organisms, including the gram-positive bacteria Staphylococcus aureus,
Streptococcus mutans and Streptococcus pyogenes (Group A strep), express
heparin-binding receptors. These heparin-binding receptors are believed to
mediate binding of organisms to heparin-like molecules in the ECM and on
host cells, e.g., endothelial cells. Heparin may also act as a bridge that
mediates adhesion of organisms to host cells that have heparin receptors.
Finally, BPI protein product may bind to gram-positive bacterial cell wall
components, such as peptidoglycans or teichoic acid, and thereby
neutralize the action of these cell wall components in inducing
gram-positive sepsis. These cell wall components are believed to play a
role in gram-positive sepsis and septic shock. Both peptidoglycans and
teichoic acids can activate the alternate complement pathway.
Gram-positive bacterial cell wall components can also elicit production of
cytokines involved in sepsis, including TNF, IL-1 and IL-6. [Bone, J.
Critical Care, 8: 51-59 (1993); Bone, Arch. Intern. Med., 154: 26-34
(1994).] Highly purified gram-positive bacterial cell wall preparations
(in which the covalent linkages and structure of peptidoglycan and
teichoic acid chains remain unaltered) have been shown to stimulate the
production of TNF-.alpha. and IL-6 by human monocytes. [Heumann et al.,
Infect. Immun., 62: 2715-2721 (1994).]
An advantage provided by the present invention is the ability to provide
more effective treatment of gram-positive bacterial infections by
improving the therapeutic effectiveness of antibiotics against
gram-positive organisms. This allows use of lower concentrations of highly
toxic or very expensive antibiotics, such as the aminoglycosides,
vancomycin, rifampin, lincomycin, chloramphenicol, and the
fluoroquinolones. Because the use of some antibiotics is limited by their
systemic toxicity or prohibitive cost, lowering the concentration of
antibiotic required for therapeutic effectiveness reduces toxicity and/or
cost of treatment, and thus allows wider use of the antibiotic. Another
advantage is the ability to treat gram-positive organisms that are
normally resistant to one or more antibiotics. The present invention may
also provide quality of life benefits due to, e.g., decreased duration of
therapy, reduced stay in intensive care units or reduced stay overall in
the hospital, with the concomitant reduced risk of serious nosocomial
(hospital-acquired) infections.
The invention also provides improved methods of in vitro decontamination of
fluids and surfaces contaminated with gram-positive bacteria by contacting
the bacteria with BPI protein product alone, or in combination with one or
more antibiotics. The amounts of BPI protein product and antibiotics used
are amounts that are separately sufficient for bactericidal/growth
inhibitory effects, or amounts sufficient to have additive or synergistic
bactericidal/growth inhibitory effects. These methods can be used in a
variety of in vitro applications including sterilization of surgical and
other medical equipment and implantable devices, including prosthetic
joints. These methods can also be used for in situ sterilization of
indwelling invasive devices such as intravenous lines and catheters, which
are often foci of infection.
The invention further provides pharmaceutical compositions for treatment of
gram-positive bacterial infection comprising BPI protein product in
combination with an antibiotic lacking gram-negative bactericidal
activity, such as lincomycin and vancomycin. The pharmaceutical
composition can optionally comprise a pharmaceutically acceptable diluent,
adjuvant or carrier. As another aspect of the invention, antiseptic
bactericidal compositions are provided which comprise a BPI protein
product alone, or in combination with an antibiotic.
"LBP protein derivatives" includes natural, synthetic and recombinantly
produced polypeptides comprising a portion of the amino acid sequence of
Lipopolysaccharide Binding Protein (LBP) holoprotein, and which are
characterized by the ability to bind to LPS but which lack the carboxy
terminal-associated immunostimulatory element(s) characteristic of the LBP
holoprotein and thus lack the CD14-mediated immunostimulatory activity
characteristic of LBP holoprotein. LBP derivative proteins are described
in detail in PCT Application No. US94/06931 filed Jun. 17, 1994, which
corresponds to co-owned, co-pending U.S. patent application Ser. No.
08/261,660, filed Jun. 17, 1994 as a continuation-in-part of U.S. patent
application Ser. No. 08/079,510, filed Jun. 17, 1993, the disclosures of
all of which are incorporated herein by reference. Preferred LBP protein
derivatives include N-terminal LBP fragments having a molecular weight of
about 25 kD or less. One LBP N-terminal fragment is characterized by the
amino acid sequence of the first 197 amino acids of the amino-terminus of
LBP set out in SEQ ID NOS: 97 and 98, and is designated LBP.sub.25. LBP
protein derivatives also include polypeptides comprising part or all of
one or more of three regions (defined by LBP amino acid sequences 17-45,
65-99 and 141-167) corresponding Coy reason of amino acid holology) to LPS
binding regions of BPI (comprising amino acid sequences 17-45, 65-99 and
142-169). LBP protein derivatives also include polypeptides comprising a
portion of the amino acid sequence of LBP holoprotein, wherein amino acids
are added, deleted, or replaced, and wherein the LBP protein derivative
maintains LPS-binding activity but lacks CD14-mediated immunostimulatory
activity. One such LBP derivative is that wherein the alaninc residue at
position 131 of the LBP.sub.25 polypeptide fragment is substituted with a
cysteine residue.
LBP derivatives include LBP derivative hybrid proteins comprising a portion
of the amino acid sequence of LBP and a portion of at least one other
polypeptide, such as BPI protein or immunoglobulin chain. One such
derivative is an LBP/BPI hybrid protein (LBP(1-197)/BPI(200-456)), which
comprises amino acid residues 1-197 of LBP followed in sequence by amino
acid residues 1-199 of BPI. Other LBP hybrid proteins comprise LBP amino
acid sequences into which all or portions of LPS binding domains of other
LPS binding proteins (such as BPI) have been inserted or substituted.
As used herein, "BPI protein product" includes naturally and recombinantly
produced BPI protein; natural, synthetic, and recombinant biologically
active polypeptide fragments of BPI protein; biologically active
polypeptide variants of BPI protein or fragments thereof, including hybrid
fusion proteins and dimers; biologically active polypeptide analogs of BPI
protein or fragments or variants thereof, including cysteine-substituted
analogs; and BPI-derived peptides. The BPI protein products administered
according to this invention may be generated and/or isolated by any means
known in the art. U.S. Pat. No. 5,198,541, the disclosure of which is
incorporated herein by reference, discloses recombinant genes encoding and
methods for expression of BPI proteins including recombinant BPI
holoprotein, referred to as rBPI.sub.50 and recombinant fragments of BPI.
Co-owned, copending U.S. patent application Ser. No. 07/885,501 and a
continuation-in-part thereof, U.S. patent application Ser. No. 08/072,063
filed May 19, 1993 and corresponding PCT Application No. 93/04752 filed
May 19, 1993, which are all incorporated herein by reference, disclose
novel methods for the purification of recombinant BPI protein products
expressed in and secreted from genetically transformed mammalian host
cells in culture and discloses how one may produce large quantities of
recombinant BPI products suitable for incorporation into stable,
homogeneous pharmaceutical preparations.
Biologically active fragments of BPI (BPI fragments) include biologically
active molecules that have the same or similar amino acid sequence as a
natural human BPI holoprotein, except that the fragment molecule lacks
amino-terminal amino acids, internal amino acids, and/or carboxy-terminal
amino acids of the holoprotein. Nonlimiting examples of such fragments
include a N-terminal fragment of natural human BPI of approximately 25 kD,
described in Ooi et al., J. Exp. Med., 1 74: 649 (1991), and the
recombinant expression product of DNA encoding N-terminal amino acids from
1 to about 193 or 199 of natural human BPI, described in Gazzano-Santoro
et al., Infect. Immun. 60: 4754-4761 (1992), and referred to as
rBPI.sub.23. In that publication, an expression vector was used as a
source of DNA encoding a recombinant expression product (rBPI.sub.23)
having the 31-residue signal sequence and the first 199 amino acids of the
N-terminus of the mature human BPI, as set out in FIG. 1 of Gray et al.,
supra, except that valine at position 151 is specified by GTG rather than
GTC and residue 185 is glutamic acid (specified by GAG) rather than lysine
(specified by AAG). Recombinant holoprotein (rBPI) has also been produced
having the sequence (SEQ ID NOS: 145 and 146) set out in FIG. 1 of Gray et
al., supra, with the exceptions noted for rBPI.sub.23 and with the
exception that residue 417 is alaninc (specified by GCT) rather than
valine (specified by GTT). Other examples include dimetic forms of BPI
fragments, as described in co-owned and co-pending U.S. patent application
Ser. No. 08/212,132, filed Mar. 11, 1994, the disclosures of which are
incorporated herein by reference. Preferred dimeric products include
dimeric BPI protein products wherein the monomers are amino-terminal BPI
fragments having the N-terminal residues from about 1 to 175 to about 1 to
199 of BPI holoprotein. A particularly preferred dimeric product is the
dimeric form of the BPI fragment having N-terminal residues 1 through 193,
designated rBPI.sub.42 dimer.
Biologically active variants of BPI (BPI variants) include but are not
limited to recombinant hybrid fusion proteins, comprising BPI holoprotein
or biologically active fragment thereof and at least a portion of at least
one other polypeptide, and dimeric forms of BPI variants. Examples of such
hybrid fusion proteins and dimeric forms are described by Theofan et al.
in co-owned, copending U.S. patent application Ser. No. 07/885,911, and a
continuation-in-part application thereof, U.S. patent application Ser. No.
08/064,693 filed May 19, 1993 and corresponding PCT Application No.
US93/04754 filed May 19, 1993, which are all incorporated herein by
reference and include hybrid fusion proteins comprising, at the
amino-terminal end, a BPI protein or a biologically active fragment
thereof and, at the carboxy-terminal end, at least one constant domain of
an immunoglobulin heavy chain or allelic variant thereof. Another example
of such a hybrid fusion protein is the recombinant expression product of
DNA encoding amino acids 1 through 199 of BPI joined to DNA encoding amino
acids 198 through 456 of LBP, designated BPI(1-199)-LBP(198-456) hybrid,
is described in PCT Application No. US94/06931 filed Jun. 17, 1994, which
corresponds to co-owned, co-pending U.S. patent application Ser. No.
08/261,660, filed Jun. 17, 1994 as a continuation-in-pan of U.S. patent
application Ser. No. 08/079,510, filed Jun. 17, 1993, the disclosures of
all of which are incorporated herein by reference.
Biologically active analogs of BPI (BPI analogs) include but are not
limited to BPI protein products wherein one or more amino acid residues
have been replaced by a different amino acid. For example, co-owned,
copending U.S. patent application Ser. No. 08/013,801 filed Feb. 2, 1993
and corresponding PCT Application No. US94/01235 filed Feb. 2, 1994, the
disclosures of which are incorporated herein by reference, discloses
polypeptide analogs of BPI and BPI fragments wherein a cysteine residue is
replaced by a different amino acid. A preferred BPI protein product
described by this application is the expression product of DNA encoding
from amino acid 1 to approximately 193 or 199 of the N-terminal amino
acids of BPI holoprotein, but wherein the cysteine at residue number 132
is substituted with alanine and is designated rBPI.sub.21 .DELTA.cys or
rBPI.sub.21. Other examples include dimetic forms of BPI analogs; e.g.
co-owned and co-pending U.S. patent application Ser. No. 08/212,132 filed
Mar. 11, 1994, the disclosures of which are incorporated herein by
reference.
Other BPI protein products useful according to the methods of the invention
are peptides derived from or based on BPI produced by recombinant or
synthetic means (BPI-derived peptides), such as those described in
co-owned and copending PCT Application No. US94/10427 filed Sep. 15, 1994,
which corresponds to U.S. patent application Ser. No. 08/306,473 filed
Sep. 15, 1994, and PCT Application No. US94/02465 filed Mar. 11, 1994,
which corresponds to U.S. patent application Ser. No. 08/209,762, filed
Mar. 11, 1994, which is a continuation-in-part of U.S. patent application
Ser. No. 08/183,222, filed Jan. 14, 1994, which is a continuation-in-part
of U.S. patent application Ser. No. 08/093,202 filed Jul. 15, 1993 (for
which the corresponding international application is PCT Application No.
US94/02401 filed Mar. 11, 1994), which is a continuation-in-part of U.S.
patent application Ser. No. 08/030,644 filed Mar. 12, 1993, the
disclosures of all of which are incorporated herein by reference.
Presently preferred BPI protein products include recombinantly-produced
N-terminal fragments of BPI, especially those having a molecular weight of
approximately between 21 to 25 kD such as rBPI.sub.23 or rBPI.sub.21, or
dimeric forms of these N-terminal fragments (e.g., rBPI.sub.42 dimer).
Additionally, preferred BPI protein products include rBPI.sub.50 and
BPI-derived peptides.
The administration of BPI protein products is preferably accomplished with
a pharmaceutical composition comprising a BPI protein product and a
pharmaceutically acceptable diluent, adjuvant, or carrier. The BPI protein
product may be administered without or in conjunction with known
surfactants, other chemotherapeutic agents or additional known
anti-microbial agents. A preferred pharmaceutical composition containing
BPI protein products (e.g., rBPI.sub.50, rBPI.sub.23) comprises the BPI
protein product at a concentration of 1 mg/ml in citrate buffered saline
(5 or 20 mM citrate, 150 mM NaCl, pH 5.0) comprising 0.1% by weight of
poloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsippany, N.J.) and 0.002%
by weight of polysorbate 80 (Tween 80, ICI Americas Inc., Wilmington,
Del.). Another preferred pharmaceutical composition containing BPI protein
products (e.g., rBPI.sub.21) comprises the BPI protein product at a
concentration of 2 mg/ml in 5 mM citrate, 150 mM NaCl, 0.2% poloxamer 188
and 0.002% polysorbate 80. Such preferred combinations are described in
co-owned, co-pending PCT Application No. US94/01239 filed Feb. 2, 1994,
which corresponds to U.S. patent application Ser. No. 08/190,869 filed
Feb. 2, 1994 and U.S. patent application Ser. No. 08/012,360 filed Feb. 2,
1993, the disclosures of all of which are incorporated herein by
reference.
Suitable antibiotics, and therapeutically effective concentrations thereof
when administered with BPI protein products, may be determined in in vivo
models or according to in vitro tests, for example, the in vitro minimum
inhibitory concentration (MIC) and in vivo mouse peritonitis or rabbit
bacteremia assays taught herein. Suitable antibiotics are antibiotics that
act on the bacterial cell wall, cell membrane, protein metabolism or
nucleic acid metabolism. These would include antibiotics or combinations
of antibiotics from the following classes: .beta.-lactam antibiotics with
or without .beta.-lactamase inhibitors, aminoglycosides, tetracyclines,
sulfonamides and trimethoprim, vancomycin, macrolides, fluoroquinolones
and quinolones, polymyxins, and other antibiotics. Dosage and
administration of suitable antibiotics are known in the art, and briefly
summarized below.
PENICILLINS
When a BPI protein product is concurrently administered with a penicillin,
for treatment of a gram-negative bacterial infection, the BPI protein
product is generally given parenterally in doses ranging from 1 .mu.g/kg
to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg to 20
mg/kg daily. The penicillin is generally given in doses ranging from 1
.mu.g/kg to 750 mg/kg daily, preferably not to exceed 24 grams daily for
adults (or 600 mg/kg daily for children), and is preferably administered
as follows:
Penicillin G is preferably administered parenterally to adults in doses
ranging from 600,000 to 1,000,000 units per day. In conventional
administration, it is effective largely against gram-positive organisms.
For treatment of pneumococcal meningitis, penicillin G is administered in
doses of 20-24 million units daily, in divided doses every 2 or 3 hours.
For children, the preferred parenteral dose of penicillin G is 300,000 to
1,000,000 units per day. One unit of penicillin G contains 0.6 .mu.g of
pure sodium penicillin G (i.e., 1 mg is 1667 units).
Amoxicillin may be administered parenterally to adults in doses ranging
from 750 mg to 1.5 grams per day, in 3 equally divided doses. For
children, preferred parenteral doses of amoxicillin range from 20 to 40
mg/kg per day in 3 equally divided doses. Amoxicillin is also available in
combination with clavulanic acid, a .beta.-lactamase inhibitor. A 250 mg
dose of the combination drug amoxicillin/clavulanate will contain 250 mg
of amoxicillin and either 125 or 62.5 mg of clavulanic acid. The
combination is preferably administered to adults orally in doses of 750 mg
per day divided into 3 equal doses every 8 hours, with a preferred dose of
1.5 grams per day for severe infections, given in 3 equally divided doses.
In children, the preferred oral dose is 20 to 40 mg/kg per day in 3
equally divided doses.
Ampicillin is preferably administered parenterally to adults in doses of 6
to 12 grams per day for severe infections, in 3 to 4 equally divided
doses. In children, the preferred parenteral dose of ampicillin is 50 to
200 mg/kg per day in 3 to 4 equally divided doses. Larger doses of up to
400 mg/kg per day, for children, or 12 grams per day, for adults, may be
administered parenterally for treatment of meningitis. Ampicillin is also
available in combination with sulbactam, a .beta.-lactamase inhibitor.
Each 1.5 gram dose of ampicillin/sulbactam contains 1 gram of ampicillin
and 0.5 grams of sulbactam. The combination is preferably administered
parenterally to adults in doses of 6 to 12 grams per day divided into 4
equal doses every 6 hours, not to exceed a total of 12 grams per day.
Azlocillin is preferably administered parenterally to adults in doses of 8
to 18 grams per day, given in 4 to 6 equally divided doses.
Carbenicillin is preferably administered parenterally to adults in doses of
30 to 40 grams per day, given by continuous infusion or in 4 to 6 equally
divided doses. Daily doses of up to 600 mg/kg have been used to treat
children with life-threatening infections.
Mezlocillin is preferably administered to adults parenterally in doses of
100 to 300 mg/kg per day, given in 4 to 6 equally divided doses. The usual
dose is 16 to 18 grams per day; for life threatening infections, 350 mg/kg
per day may be administered, but in doses not to exceed 24 grams per day
given in 6 equally divided doses every 4 hours. For children, the
preferred parenteral dose of mezlocillin is 150 to 300 mg/kg per day.
Nafcillin is preferably administered intravenously to adults in doses of 3
grams per day, given in 6 equally divided doses every 4 hours, with
doubled doses for very severe infections. In conventional administration,
it is effective largely against gram-positive organisms. In children, the
preferred parenteral dose is 20 to 50 mg/kg per day, in 2 equally divided
doses every 12 hours. The preferred oral dose for nafcillin ranges from 1
gram per day to 6 grams per day in 4 to 6 divided doses.
Oxacillin is preferably administered parenterally to adults in doses of 2
to 12 grams per day, in 4 to 6 equally divided doses. In conventional
administration, it is effective largely against gram-positive organisms.
In children, oxacillin is preferably administered in doses of 100 to 300
mg/kg per day.
Piperacillin is preferably administered parenterally to adults in doses
ranging from 100 mg/kg, or 6 grams per day, in 2 to 4 equally divided
doses, up to a maximum of 24 grams per day, in 4 to 6 equally divided
doses. Higher doses have been used without serious adverse effects.
Ticarcillin is preferably administered parenterally to adults in doses
ranging from 4 grams per day to 18 grams per day administered in 4 to 6
equally divided doses. The usual dose is 200 to 300 mg/kg per day. For
children, the preferred parenteral dose of ticarcillin ranges from 50
mg/kg per day to 300 mg/kg per day, given in 3, 4 or 6 equally divided
doses. The combination ticarcillin/clavulanate is preferably administered
parenterally to adults in doses of 200 to 300 mg/kg per day (based on
ticarcillin content), in 4 to 6 equally divided doses. For adults, the
usual dose is 3.1 grams (which contains 3 grams of ticarcillin and 100 mg
of clavulanic acid) every 4 to 6 hours. The combination is also available
in a dose of 3.2 grams, which contains 3 grams of ticarcillin and 200 mg
of clavulanic acid.
In general, it is desirable to limit each intramuscular injection of a
penicillin or cephalosporin to 2 grams; larger doses should be
administered by multiple injections in different large muscle masses.
CEPHALOSPORINS
When a BPI protein product is concurrently administered with a
cephalosporin, for treatment of a gram-negative bacterial infection, the
BPI protein product is generally given parenterally in doses ranging from
1 .mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1
mg/kg to 20 mg/kg daily. The cephalosporin is generally given in doses
ranging from 1 .mu.g/kg to 500 mg/kg daily, preferably not to exceed 16
grams daily, and is preferably administered as follows:
Cefamandole is preferably administered parenterally to adults in doses
ranging from 1.5 grams per day, given in 3 equally divided doses every 8
hours, to 12 grams per day for life-threatening infections, given in 6
equally divided doses every 4 hours. In children, cefamandole is
preferably administered in doses ranging from 50 to 150 mg/kg per day, in
3 to 6 equally divided doses, not to exceed a total of 12 grams per day.
Cefazolin is preferably administered parenterally to adults in doses of 750
mg per day, given in 3 equally divided doses every 8 hours. In severe,
life-threatening infections, it may be administered at doses of 6 grams
per day divided into 4 equal doses every 6 hours; in rare instances, up to
12 grams per day have been used. In children, the preferred parenteral
dose of cefazolin is 20 to 50 mg/kg per day, divided into 3 or 4 equal
doses, with 100 mg/kg per day administered for severe infections.
Cefonicid is preferably administered parenterally to adults in doses
ranging from 500 mg once daily, to 2 grams once daily for life-threatening
infections. For intramuscular administration, a 2 gram dose should be
divided into two 1-gram injections.
Cefoperazone is preferably administered parenterally to adults in doses
ranging from 2 grams per day, given in 2 equally divided doses every 12
hours, to 12 grams per day for severe infections, given in 2, 3 or 4
equally divided doses. Doses up to 16 grams per day have been administered
without complications.
Cefotetan is preferably administered parenterally to adults in doses of 1
to 4 grams per day, in 2 equally divided doses every 12 hours. Cefotetan
may be administered in higher doses for fife-threatening infections, not
to exceed a total dose of 6 grams per day.
Cefotaxime is preferably administered parenterally to adults in doses
ranging from I to 12 grams per day, not to exceed 12 grams per day (2
grams every 4 hours) for fife-threatening infections. In children, the
parenteral dose of cefotaxime is preferably 50 to 180 mg/kg, divided into
4 to 6 equal doses.
Cefoxitin is preferably administered parenterally to adults in doses
ranging from 3 to 12 grams per day, given in 3, 4, or 6 equally divided
doses. In children, cefoxitin is preferably administered parenterally in
doses of 80 to 160 mg/kg per day, given in 4 or 6 equally divided doses,
not to exceed a total dose of 12 grams per day.
Ceftazidime is preferably administered parenterally to adults in doses
ranging from 500 mg per day, given in 2 to 3 equally divided doses (every
8 or 12 hours), up to a maximum of 6 grams per day. In children,
ceftazidime is preferably administered intravenously in doses of 30 to 50
mg/kg, to a maximum of 6 grams per day.
Ceftizoxime is preferably administered parenterally to adults in doses
ranging from 1 gram per day, given in 2 equally divided doses every 12
hours, to 12 grams per day for life-threatening infections, given in 3
equally divided doses every 8 hours. The usual adult dose is 1 to 2 grams
every 8 or 12 hours. For children, the preferred parenteral dose is 50
mg/kg every 6 or 8 hours, for a total daily dose of 200 mg/kg.
Ceftriaxone is preferably administered parenterally to adults in doses
ranging from 1 to 2 grams per day, given in 2 equally divided doses every
12 hours. It may be given in higher doses, not to exceed a total of 4
grams per day. In children, the preferred parenteral dose of ceftriaxone
is 50 to 75 mg/kg per day, not to exceed 2 grams per day. In meningitis,
ceftriaxone may be administered in doses of 100 mg/kg per day, not to
exceed 4 grams per day.
Cefuroxime is preferably administered parenterally to adults in doses
ranging from 2.25 to 4.5 grams per day, in 3 equally divided doses every 8
hours. For life-threatening infections, 6 grams per day may be
administered in 4 equally divided doses every 6 hours, and for meningitis,
9 grams per day may be administered in 3 equally divided doses every 8
hours. For children, the preferred parenteral dose of cefuroxime is 50 to
150 mg/kg per day in 3 to 4 equally divided doses, or 240 mg/kg per day
for meningitis.
Cephalexin is formulated for oral administration, and is preferably
administered orally to adults in doses ranging from 1 to 4 grams per day
in 2 to 4 equally divided doses. For children, the preferred dose is 20 to
50 mg/kg per day in divided doses, with doses being doubled for severe
infections.
Cephalothin is usually administered parenterally to adults in doses of 8 to
12 grams per day.
OTHER BETA-LACTAMS
When a BPI protein product is concurrently administered with an imipenem
antibiotic, for treatment of a gram-negative bacterial infection, the BPI
protein product is generally given parenterally in doses ranging from 1
.mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg
to 20 mg/kg daily. The imipenem is generally given in doses ranging from 1
.mu.g/kg to 100 mg/kg daily, and is preferably administered as follows:
Imipenem is available in combination with cilastin, an inhibitor of the
renal dipeptidase enzyme that rapidly inactivates imipenem. The
combination is preferably administered intramuscularly to adults in doses
of 1 to 1.5 grams per day, given in 2 equally divided doses every 12
hours. Intramuscular doses exceeding 1.5 grams per day are not
recommended. The combination is preferably administered intravenously in
doses ranging from 1 to 4 grams per day, in 4 equally divided doses every
6 hours; doses exceeding 50 mg/kg per day, or 4 grams per day, are not
recommended.
When a BPI protein product is concurrently administered with a monobactam
antibiotic, for treatment of a gram-negative bacterial infection, the BPI
protein product is generally given parenterally in doses ranging from 1
.mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg
to 20 mg/kg daily. The monobactam is generally given in doses ranging from
1 .mu.g/kg to 200 mg/kg daily, and is preferably administered as follows:
Aztreonam is preferably administered parenterally to adults in doses
ranging from 1 gram per day, given in 2 equally divided doses every 12
hours, up to a maximum recommended dose of 8 grams per day in cases of
life-threatening infection, given in 3 or 4 equally divided doses.
AMINOGLYCOSIDES
When a BPI protein product is concurrently administered with an
aminoglycoside, for treatment of a gram-negative bacterial infection, the
BPI protein product is generally given parenterally in doses ranging from
1 .mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1
mg/kg to 20 mg/kg daily. The aminoglycoside is generally given in doses
ranging from 1 .mu.g/kg to 20 mg/kg daily, preferably not to exceed 15
mg/kg daily, and is preferably administered as follows:
When administering aminoglycosides, it is desirable to measure serum peak
and trough concentrations to ensure the adequacy and safety of the dosage.
Dosages should generally be adjusted to avoid toxic peak and trough
concentrations. Amikacin is preferably administered parenterally to adults
and children in doses of 15 mg/kg per day, divided into two or three equal
doses every 8 or 12 hours, and not to exceed a total dose of 1.5 grams per
day. For uncomplicated infections, a dose of 500 mg amikacin per day, in 2
equally divided doses, may be administered. Dosages should be adjusted to
avoid prolonged serum peak concentrations of amikacin above 35 .mu.g/ml
and prolonged trough concentrations greater than 10 .mu.g/ml.
Gentamicin is preferably administered parenterally to adults in doses of 3
mg/kg per day, in three equally divided doses every 8 hours. For
life-threatening infections, up to 5 mg/kg per day in 3 to 4 equally
divided doses may be administered, but this dosage should be reduced to 3
mg/kg per day as soon as clinically indicated. For children, gentamicin is
preferably administered parenterally in doses of 6 to 7.5 mg/kg per day.
Dosages should be adjusted to avoid prolonged serum peak concentrations of
gentamicin above 12 .mu.g/ml and prolonged trough concentrations greater
than 2 .mu.g/ml.
Netilmicin may be administered parenterally to adults in doses ranging from
3 mg/kg per day, in 2 equally divided doses every 12 hours, to 6.5 mg/kg
per day for serious systemic infection, in 2 or 3 equally divided doses.
In children, the preferred parenteral dose is 5.5 to 8 mg/kg per day, in 2
or 3 equally divided doses. Dosages should be adjusted to avoid prolonged
serum peak concentrations of netilmicin above 16 .mu.g/ml and prolonged
serum trough concentrations above 4 .mu.g/ml.
Tobramycin is preferably administered parenterally to adults in doses of 3
mg/kg per day, given in three equally divided doses every 8 hours. For
life-threatening infections, tobramycin may be administered in doses up to
5 mg/kg per day, in 3 or 4 equally divided doses, but this dosage should
be reduced to 3 mg/kg per day as soon as clinically indicated. In
children, tobramycin is preferably administered parenterally in doses of 6
to 7.5 mg/kg per day. Prolonged serum concentrations of tobramycin above
12 .mu.g/ml should be avoided, and rising trough levels above 2 .mu.g/ml
may indicate tissue accumulation, which may contribute to toxicity.
Concurrent administration of BPI protein product with the aminoglycosides,
including amikacin, gentamicin, netilmicin and tobramycin, may permit a
lowering of the dose of these toxic antibiotics necessary to achieve a
therapeutic effect.
TETRACYCLINES
When a BPI protein product is concurrently administered with a
tetracycline, for treatment of a gram-negative bacterial infection, the
BPI protein product is generally given parenterally in doses ranging from
1 .mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1
mg/kg to 20 mg/kg daily. The tetracycline is generally given in doses
ranging from 1 .mu.g/kg to 50 mg/kg daily, and is preferably administered
as follows:
The tetracycline antibiotics are generally administered to adults in doses
of 1 to 2 grams per day. An exception is doxycycline, which is preferably
administered intravenously to adults in doses of 100 to 200 mg per day,
and to children in doses of 2 mg/lb per day. Tetracycline may be
administered parenterally to adults in doses of 0.5 to 2 grams per day, in
2 equally divided doses, and to children in doses of 10 to 20 mg/kg per
day.
SULFONAMIDES
When a BPI protein product is concurrently administered with a sulfonamide
or trimethoprim, for treatment of a gram-negative bacterial infection, the
BPI protein product is generally given parenterally in doses ranging from
1 .mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1
mg/kg to 20 mg/kg daily. The sulfonamide or trimethoprim is generally
given in doses ranging from 1 .mu.g/kg to 150 mg/kg daily, preferably not
to exceed a combination dose of 960 mg trimethoprim/4.8 g sulfamethoxazole
daily, and is preferably administered as follows:
The combination trimethoprim/sulfamethoxazole is available in a formulation
containing a 1: 5 ratio of trimethoprim and sulfamethoxazole (e.g., 16 mg
trimethoprim and 80 mg sulfamethoxazole). The combination is preferably
administered intravenously to adults or children in doses of 8 to 10 mg/kg
(based on the weight of the trimethoprim component) per day, in 2 to 4
equally divided doses. For Pneumocystis carinii infection, the combination
can be administered in doses of 20 mg/kg (based on the weight of the
trimethoprim component) per day, in 3-4 equally divided doses, to a
maximum recommended dose of 960 mg trimethoprim/4.8 g sulfamethoxazole per
day. Trimethoprim alone is preferably administered orally to adults in
doses of 200 mg per day. Sulfamethoxazole alone is preferably administered
orally to adults in doses of 2 to 3 grams per day, and to children orally
in doses of 50 to 60 mg/kg per day.
FLUOROQUINOLONES
When a BPI protein product is concurrently administered with a
fluoroquinolone or quinolone, for treatment of a gram-negative bacterial
infection, the BPI protein product is generally given parenterally in
doses ranging from 1 .mu.g/kg to 100 mg/kg daily, and preferably at doses
ranging from 1 mg/kg to 20 mg/kg daily. The fluoroquinolone or quinolone
is generally given in doses ranging from 1 .mu.g/kg to 50 mg/kg daily,
preferably not to exceed 1 gram daily, and is preferably administered as
follows:
Norfloxacin is preferably administered orally to adults in doses from 400
to 800 mg daily, divided into two doses every 12 hours. Cinoxacin is
preferably administered orally to adults in doses of 1 gram per day, given
in 2 or 4 equally divided doses. Ciprofloxacin is preferably administered
to adults intravenously in doses from 400 to 800 mg daily, or orally in
doses from 500 to 1500 mg daily, divided into two doses every 12 hours.
Ofloxacin is preferably administered to adults intravenously in doses from
400 to 800 mg daily, or orally in doses from 400 to 800 mg daily, divided
into two doses every 12 hours.
VANCOMYCIN
When a BPI protein product is concurrently administered with vancomycin,
for treatment of a gram-negative bacterial infection, the BPI protein
product is generally given parenterally in doses ranging from 1 .mu.g/kg
to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg to 20
mg/kg daily. The vancomycin is generally given in doses ranging from 1
mg/kg to 50 mg/kg daily, and is preferably administered parenterally to
adults in doses of 2 grams per day, divided into 2 or 4 doses every 6 or
12 hours. In children it is preferably administered in doses of 40 mg/kg,
given in 4 equally divided doses every 6 hours. In conventional
administration, vancomycin is effective largely against gram-positive
organisms.
MACROLIDES
When a BPI protein product is concurrently administered with a macrolide,
for treatment of a gram-negative bacterial infection, the BPI protein
product is generally given parenterally in doses ranging from 1 .mu.g/kg
to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg to 20
mg/kg daily. The macrolide is generally given in doses ranging from 1
.mu.g/kg to 100 mg/kg daily, and is preferably administered as follows:
Erythromycin is preferably administered intravenously to adults and
children in doses of 15 to 20 mg/kg per day, given by continuous infusion
or in 4 equally divided doses every 6 hours. Erythromycin can be
administered at doses up to 4 grams per day in cases of very severe
infection.
Clarithromycin is preferably administered orally to adults in doses of 500
mg to 1 gram daily, in equally divided doses every 12 hours.
Azithromycin is preferably administered orally to adults at a dose of 500
mg on the first day of treatment followed by 250 mg once daily for 4 days,
for a total dose of 1.5 grams.
OTHERS
When a BPI protein product is concurrently administered with other
antibiotics, for treatment of a gram-negative bacterial infection, the BPI
protein product is generally given parenterally in doses ranging from 1
.mu.g/kg to 100 mg/kg daily, and preferably at doses ranging from 1 mg/kg
to 20 mg/kg daily.
Polymyxin B is generally given in doses ranging from 1 unit/kg to 45,000
units/kg daily, and is preferably administered intravenously to adults and
children in doses of 15,000 to 25,000 units/kg per day, divided into 2
equal doses every 12 hours. It may be administered intramuscularly in
doses of 25,000 to 30,000 units/kg per day, although these injections are
very painful. Doses of polymyxin B as high as 45,000 units/kg per day have
been used in limited clinical studies to treat neonates for Pseudomonas
aeruginosa sepsis. Polymyxin B is the treatment of choice for P.
aeruginosa meningitis, and is preferably administered intrathecally to
adults and older children in doses of 50,000 units once daily for to 4
days, followed by 50,000 units every other day; in children under two
years old, it is administered intrathecally in doses of 20,000 daily for 3
to 4 days, followed by 25,000 units every other day.
Chloramphenicol is preferably administered intravenously to adults in doses
of 50 mg/kg per day, in 4 equally divided doses; in exceptional cases, it
can be administered in doses up to 100 mg/kg per day. In children,
chloramphenicol is preferably administered intravenously in doses of 25
mg/kg per day, although up to 100 mg/kg per day can be administered in
cases of severe infection.
Clindamycin is preferably administered parenterally to adults in doses
ranging from 600 mg to 4.8 grams per day, given in 2, 3 or 4 equally
divided doses. It is recommended that the dose in each intramuscular
injection not exceed 600 mg. For children, clindamycin is preferably
administered parenterally in doses of 15-40 mg/kg per day, given in 3 or 4
equally divided doses.
Dosages of all antimicrobial agents should be adjusted in patients with
renal impairment or hepatic insufficiency, due to the reduced metabolism
and/or excretion of the drugs in patients with these conditions. Doses in
children should also be reduced, generally according to body weight. Those
skilled in the art can readily optimize effective dosages and
administration regimens for the BPI protein product and the antibiotics in
concurrent administration.
Other aspects and advantages of the present invention will be understood
upon consideration of the following illustrative examples. Example 1
addresses plate assays of the bactericidal effect in vitro of a BPI
protein product on the gram-positive organism Bacillus subtilis. Example 2
relates to plate assays of the in vitro growth inhibitory effect of a BPI
protein product on the gram-positive organism Staphylococcus aureus and
its L-phase variant. Example 3 evaluates the in vitro bactericidal and
growth inhibitory effects of a variety of BPI protein products on
Staphylococcus aureus, penicillin-treated S. aureus, and the L-phase
variant, as measured in radial diffusion assays, plate assays, protein
synthesis assays, and broth growth inhibition assays. Examples 4 and 5
evaluate the in vitro growth inhibitory effects of a variety of BPI
protein products on L-phase variants of Streptococcus pneumoniae and
Enterococcus faecalis, respectively. Example 6 examines the effect of a
variety of BPI protein products on the mycoplasma Acholeplasma laidlawii.
Example 7 examines the in vitro growth inhibitory effect of an LBP-BPI
hybrid on a variety of gram-positive L-phase variants. Example 8 relates
to radial diffusion assays measuring the growth inhibitory effect of a
variety of BPI synthetic peptides on S. aureus. Examples 9-17 address
large-scale screening of the bactericidal/growth inhibitory effect and the
antibiotic susceptibility-increasing effect of BPI protein products on a
variety of gram-positive organisms: S. pneumoniae (Example 9),
Streptococcus pyogenes (Group A strep) (Example 10), Streptococcus
agalactia (Group B strep) (Example 11), Streptococcus bovis (Example 12),
E. faecalis (Example 13), Enterococcus faecium (Example 14), a variety of
Enterococcus species (Example 15), S. aureus (Example 16), Staphylococcus
epidermidis (Example 17), and various coagulase-negative Staphylococcus
species (Example 18). Example 19 examines the early in vitro bactericidal
effect of BPI and selected antibiotics on Streptococcus pneumoniae,
Staphylococcus aureus and Enterococcus faecalis. Example 20 evaluates the
effect of a BPI protein product, alone or with antibiotics, on
Streptococcus pneumoniae (especially penicillin-resistant strains),
Staphylococcus aureus (especially methicillin-resistant strains),
Enterococcus (especially multiply resistant strains), and Corynebacteria.
Example 22 evaluates the in vivo effects of BPI protein products alone or
in combination with antibiotics in animal models.
EXAMPLE 1
IN VITRO BACTERICIDAL EFFECTS OF A BPI PROTEIN PRODUCT ON BACILLUS SUBTILIS
A plate assay was used to determine the effects of a BPI protein product on
the gram-positive organism Bacillus subtilis (ATCC Accession No. 6633).
The cells were incubated overnight at 37.degree. C. in Brain Heart
Infusion (BHI) broth (Difco, Detroit, Mich.), after which the optical
density of the cell suspension was adjusted with additional BHI broth to
A.sub.600 =.about.1.0 (equivalent to about 5.times.10.sup.8 CFU/ml). The
cells were washed twice with 0.9% NaCl and resuspended in D-PBS, pH 7.4,
to a cell density of approximately 1-2.times.10.sup.4 CFU/ml. The cells
were diluted at a ratio of 1: 10 in Eafi's MEM medium (GIBCO, Grand
Island, N.Y.) and 190 .mu.l of the diluted cells (200-400 cells per well)
added to low protein-binding 96 well plates (Coming, N.Y.). The
rBPI.sub.23 at varying concentrations was added to the wells to achieve
final concentrations ranging from 200 to 1,000 nM in a total volume of 200
.mu.l in each well, and the plates were incubated at 37.degree. C. for 30
minutes or 60 minutes. After incubation, 100 .mu.l from each well was
plated on brain heart infusion agar plates. After 24 hours of incubation
at 37.degree. C., the number of colonies on each agar plate was counted.
In multiple experiments, rBPI.sub.23 displayed clear and reproducible
bactericidal activity at concentrations as low as 200 nM (about 4.5
.mu.g/ml). The results of a representative experiment are displayed in
FIG. 1, which shows that the bactericidal effects of rBPI.sub.23 were
dose-dependent. The bactericidal effects of rBPI.sub.23 were more
pronounced after 60 minutes of incubation than after 30 minutes.
EXAMPLE 2
IN VITRO EFFECTS OF A BPI PROTEIN PRODUCT ON STAPHYLOCOCCUS AUREUS AND ITS
L-PHASE VARIANT IN A PLATE ASSAY
A plate assay was conducted to test the effect of a BPI protein product on
the gram-positive organism Staphylococcus aureus (ATCC Accession No.
19636) and its L-phase variant (ATCC Accession No. 19640). An L-phase
variant (also called an L-form) lacks a cell wall and must be grown in the
presence of osmotically protective media containing serum (usually 5 to
10% horse serum).
S. aureus, or Escherichia coli J5 as a control were grown overnight in
heart infusion (HI) broth and tryptone yeast extract (TYE) broth (both
broths from Difco, Detroit, Mich.), respectively, then diluted 1: 100 in
the same broth and grown to mid-log phase (A.sub.600 =.about.0.5). The S.
aureus L-phase variant cells were grown on agar plates containing HI broth
supplemented with 3.5% NaCl and 10% horse serum, and colonies were
resuspended in 4 ml of HI broth supplemented with 3.5% NaCl to an
A.sub.600 of approximately 0.2 to 0.5. The cell suspensions were spread on
agar plates containing HI broth supplemented with 3.5% NaCl and 5% horse
serum. rBPI.sub.21 was spotted on the surface of the plates in amounts
varying from 0.75 to 3 .mu.g, in 2 .mu.l. No growth inhibition zones were
observed on the S. aureus plates, but for the L-phase variant, a clear
zone of growth inhibition was observed at 3 .mu.g rBPI.sub.21, and partial
clear zones at 1.5 and 0.75 .mu.g rBPI.sub.21. In the control E. coli
plate, clear zones were observed at 1.0 and 0.5 of rBPI.sub.21. No zones
of growth inhibition were observed for plates on which buffer alone was
spotted. The results indicate that in the absence of a cell wall, growth
of the gram-positive bacterium S. aureus is affected by BPI protein
products.
EXAMPLE 3
IN VITRO EFFECTS OF VARIOUS BPI PROTEIN PRODUCTS ON STAPHYLOCOCCUS AUREUS,
PENICILLIN-TREATED S. AUREUS, AND S. AUREUS L-PHASE VARIANT
A. EVALUATION IN RADIAL DIFFUSION ASSAYS
Radial diffusion assays were performed to determine the effect of various
BPI protein products on S. aureus (ATCC Accession No. 19636) and
penicillin-treated S. aureus or natural L-phase variants (ATCC Accession
No. 19640) grown in osmotically protective media. Because penicillin
inhibits peptidoglycan synthesis, cells that have been contacted with
penicillin lose their cell walls once growth is initiated. If osmotic
protection is provided, a certain percentage of the cells survive and grow
as L-phase variants. This example was directed towards determining whether
penicillin-treated cells or L-phase variants would be susceptible to BPI
protein products because the protective barrier of the cell wall was
absent.
S. aureus cells were grown overnight to mid-log phase in HI broth and were
adjusted using the same broth to an optical density of A.sub.600
=.about.0.25 (equivalent to about 2.5.times.10.sup.8 cells/ml) or 2.5
(equivalent to about 2.5.times.10.sup.9 cells/ml). 80 .mu.l of the cell
suspension was added to 8 ml of molten agar medium containing 0.8%
agarose, HI broth supplemented with 3.5% NaCl, and 1,000 units/ml
penicillin G (Sigma, St. Louis, Mo.). The agar was poured into 90 mm
plates and allowed to solidify. As a control, the same S. aureus cells
were adjusted to an optical density of A.sub.600 =0.25, 0.025, 0.0025 or
0.00025, and added to the same agar medium, but without penicillin. Holes,
or wells, of 3 mm diameter were prepared in the 90 mm plates, and 5 .mu.l
of two-fold serial dilutions of rBPI.sub.21 were added to each well. For
the penicillin-treated cells, 240, 120, 60, 30, 15 and 7.5 picomoles
(pmol) per well of rBPI.sub.21 were tested, and the plates were incubated
for 48 hours at 37.degree. C. in a CO.sub.2 incubator. For the control
cells (not grown on penicillin G-containing plates), 400, 200, 100 and 50
pmol/well of rBPI.sub.21 were tested, and the plates were incubated for 24
hours. The plates were then examined for zones of inhibition. The results,
shown below in FIG. 2, demonstrate that the BPI protein product inhibited
bacterial growth in a dose-dependent manner; rBPI.sub.21 at levels as low
as 30 pmol per well was able to inhibit growth of the penicillin-treated
S. aureus cells. In contrast, even the highest concentration of
rBPI.sub.21 (400 pmol) had no effect on the control S. aureus cells grown
without penicillin.
These results show that, while the normal bacterial form of S. aureus is
not susceptible to BPI protein products, it can be rendered susceptible by
penicillin treatment that causes loss of the cell wall due to inhibition
of cell wall synthesis. This indicates that gram-positive bacteria are
susceptible to BPI protein products if the cell wall is removed and the
cytoplasmic membrane is exposed.
Additional radial diffusion assays were conducted on the natural S. aureus
L-phase variant to confirm the above results. The cells were grown
overnight in HI broth supplemented with 3.5% NaCl 10 mM CaCl.sub.2 and
1000 units/ml penicillin G, to an A.sub.600 =.about.0.3. The cells were
used either undiluted or as a 1: 10 dilution. 80 .mu.l of the cell
suspension was added to 8 ml of molten HI agar medium containing HI broth
supplemented with 3.5% NaCl and 0.8% agarose. The agar was poured into 90
mm plates and allowed to solidify. As a control, a plate using the same
medium was also prepared using 80 .mu.l of normal S. aureus cells adjusted
to A.sub.600 =2.5 with HI broth supplemented with 3.5% NaCl, 10 mM CaCl,
and 1000 .mu./ml penicillin G. Holes, or wells, of 3 mm diameter were
prepared in the 90 mm plates, and 5 .mu.l of rBPI.sub.21 in two-fold
serial dilutions, starting at 5 .mu.g/5 .mu.l (or 240 pmol/5 .mu.l), was
added to the wells. The plates were incubated for 48 hours and then
examined for zones of growth inhibition. The results of this experiment,
shown in FIG. 3, demonstrate that the L-phase variant is also susceptible
to BPI protein products.
Further radial diffusion assays were conducted to evaluate the effect of
various BPI protein products, rBPI.sub.23, rBPI.sub.21, rBPI.sub.50,
BPI-Immunoglobulin fusion (the expression product of 1.c."p"ING4512
described in Theofan, et al., U.S. Application No. 07/885,911 filed May
19, 1992 and 08/064,693 filed May 19, 1993) and rBPI.sub.42 dimer (the
dimeric form of N-terminal (1-193) BPI, described in Amons, et al., U.S.
Application No. 08/212,132 filed Mar. 11, 1994) on penicillin-treated S.
aureus. S. aureus were grown to log phase in HI broth supplemented with
3.5% NaCl, 5 mM CaCl.sub.2 and 1000 units/ml penicillin G, and was
adjusted to A.sub.600 =.about.0.025. The cell suspension was added in a 1:
100 dilution to molten HI agar medium containing HI broth supplemented
with 3.5% NaCl, 0.8% agarose, and 1000 units/ml penicillin G, which was
then allowed to solidify. Serial 2-fold dilutions of each BPI protein
product in 5 .mu.l were added to 3-mm wells prepared in the plates. After
24 hours of incubation, the plates were examined for zones of growth
inhibition. The results of these assays, shown in FIG. 4, demonstrate that
these BPI protein products are all effective in inhibiting growth of
penicillin-treated S. aureus, with BPI-Immunoglobulin fusion and
rBPI.sub.42 dimer being the most potent.
When these experiments were repeated using other species of bacteria,
Staphylococcus epidermidis (ATCC Accession No. 35983), Enterococcus
faecalis (ATCC Accession No. 4200), and Streptococcus pneumoniae (ATCC
Accession No. 35088), using the BPI protein products rBPI.sub.23 (5
.mu.g/well), rBPI.sub.21 (10 .mu.g/well), rBPI42 dimer (5 .mu.g/well),
rBPI.sub.50 (20 .mu.g/well) and rBPI-Ig fusion (5 .mu.g/well), no growth
inhibition was observed for any of these BPI protein products. These
results indicate that in this assay, the normal bacterial forms of these
species of bacteria are also not susceptible to the BPI protein products
when used at the concentrations indicated. Higher concentrations of these
BPI protein products may produce different susceptibility results.
The effects of additional BPI protein products, BPI-derived peptides, on
the S. aureus bacterial form (ATCC No. 19636) and its corresponding
L-phase variant (ATCC No. 19640) were also evaluated in this radial
diffusion assay. The cells were grown to log phase in either HI broth (for
the bacterial forms) or HI broth supplemented with 3.5% NaCl, 5 mM
CaCl.sub.2 and 1000 U/mL penicillin G (for the L-phase variants). rBPI42
dimer and the BPI-derived peptides XMP. 13, XMP.30, XMP.48 and XMP. 102
(as described in Example 8 and Table 1 infra) were dissolved at .about.2
mg/mL in PBS (without calcium and magnesium) and the corrected
concentration determined by comparing absorbance at 280 nM with that
predicted by the extinction co-efficient. The bacteria were incorporated
into agarose, 5 .mu.l aliquots of various concentrations of the peptides
or rBPI.sub.42 dimer were added to each well, and the plates were
incubated at 37.degree. C for 24 hours (bacterial form) or 48 hours
(L-phase variants). The results, shown in FIGS. 5 and 6, indicate that
XMP.48 was active against the bacterial form and that all of the peptides
tested were active against the L-phase variants. The rBPI42 dimer appeared
to be about ten-fold more active on a molar basis than any of the
peptides, while XMP. 13 appeared to be the least potent.
The experiment was repeated with BPI-derived peptides XMP. 14, XMP.2, XMP.
13, XMP.30, XMP.48 and XMP. 102 on the L-phase variant and
penicillin-treated bacterial form of S. aureus. The results, shown in
FIGS. 7 and 8, establish that XMP. 14 was completely inactive against the
L-phase variant and XMP.2 was less active than XMP. 13 on both the L-phase
variant and the penicillin-treated bacterial form.
B. EVALUATION IN A PLATE ASSAY AND A PROTEIN SYNTHESIS ASSAY WITH S. AUREUS
L-PHASE VARIANT
Plate assays were performed to confirm the relative potency of rBPI.sub.42
dimer and examine the time course of its bactericidal effects. Cells were
grown to A.sub.600 =0.3 (.about.3.times.10.sup.8 cells/mL) in HI broth
supplemented with NaCl to 0.4M and 5 mM CaCl.sub.2, diluted 1: 100 into
NaCl-supplemented PBS, incubated with rBPI.sub.21, rBPI.sub.50,
rBPI.sub.42 dimer or buffer, and plated on HI agar supplemented with NaCl
and 1% horse serum. The results showed that rBPI.sub.42 dimer caused up to
a 75% reduction in colony forming units (CFUs) relative to buffer-treated
cells at 2 hours. Incubation of cells with either buffer or 50 .mu.g/mL
rBPI.sub.42 dimer for up to 5 hours before being plated produced optimal
killing by dimer at 3 hours. In these assays, rBPI.sub.42 dimer was
consistently and significantly more potent than either of the monomeric
forms, rBPI.sub.50 or rBPI.sub.21. To further extend these studies, the
effect of these BPI protein products on protein synthesis of the S. aureus
L-phase variant was examined. In these experiments, cells were grown to
A.sub.600 =0.5 (.about.5.times.10.sup.8 cells/mL) in HI broth supplemented
with NaCl to 0.4M and 5 mM CaCl.sub.2, diluted 1: 100 into
NaCl-supplemented HI broth and incubated with BPI protein product.
Following incubation at 37.degree. C., 0.4 .mu.L of .sup.14 C-amino acids
was added and incubation was continued for 30 min. at 37.degree. C.
Incorporation of the .sup.14 C-amino acids was linear for at least 30 min.
Cells were treated with 3 mL cold 10% TCA to arrest protein synthesis and
release free .sup.14 C-amino acids from the cells. The cells were then
applied to 0.45 .mu.m-pore size HA Millipore filters (Millipore, Bedford,
Mass.), washed once with 3 mL 10% TCA and then with 5 mL water. The
filters were dried and counted, and the percentage of .sup.14 C-amino
acids incorporated relative to the control was calculated. An initial
experiment to establish the optimum incubation time with 50 .mu.g/mL
rBPI.sub.42 dimer demonstrated that maximal inhibition of protein
synthesis (.about.90%) was achieved by 2 hours. Incubation of cells for 2
hours with various concentrations of BPI protein product and then with
.sup.14 C-amino acids demonstrated that, as in other assays, rBPI.sub.42
dimer was significantly more potent than the other BPI protein products.
The rBPI.sub.21 appeared to be less potent than rBPI.sub.23 or rBPI.sub.50
at inhibiting protein synthesis. Thus, patterns of growth inhibition
parallel the patterns of inhibition of protein synthesis, with rBPI.sub.42
dimer being most potent at inhibiting growth and protein synthesis.
C. EVALUATION IN A BROTH GROWTH INHIBITION ASSAY WITH S. AUREUS L-PHASE
VARIANT
Broth growth inhibition assays were performed to determine the effect of a
BPI protein product on the growth of S. aureus L-phase variant cells (ATCC
Accession No. 19640). Initial attempts to grow L-phase variant cells in HI
broth supplemented with horse serum resulted in poor growth or dumping.
The effect of CaCl.sub.2 concentration on the growth of L-phase variant
cells was tested to determine whether the cells could be readily grown in
media supplemented with CaCl.sub.2 instead of horse serum. L-phase variant
cells were inoculated from an overnight culture into 2 ml of HI broth
supplemented with 3.5% NaCl, 1,000 units/ml penicillin G and various
concentrations of calcium chloride. The cells were incubated at 37.degree.
C. on a rotary shaker and the optical density (A.sub.600) was measured at
set time points. The results, depicted in FIG. 9, show that 5-10 mM
CaCl.sub.2 maintains an optimal rate of cell growth. These results show
that CaCl.sub.2 can be substituted for serum to allow growth of the
L-phase variants in broth.
L-phase variant cells of S. aureus were cultured overnight in HI broth
supplemented with 3.5% NaCl, 10 mm CaCl.sub.2 and 1,000 units/ml
penicillin G. The cells were adjusted to an optical density of A.sub.600
=.about.0.025 (equivalent to .about.2.5.times.10.sup.7 cells/ml) in the
same broth and 150 .mu.l of cells was added to each well of a 96 well
plate. Varying concentrations of rBPI.sub.21, diluted in buffer containing
5 mM citrate and 150 mM NaCl to a total volume of 2.5 .mu.l, were added to
the wells. Control wells contained cells with dilution buffer only (no BPI
protein product) or with 16 .mu.g/ml lipopolysaccharide binding protein
(LBP). The cells were incubated at 37.degree. C. on a rotary shaker and
cell growth (A.sub.600) was followed over 7 hours. The results are shown
in FIG. 10, which depicts the change in A.sub.600 over time for varying
concentrations of rBPI.sub.21, LBP or control with no BPI protein product.
The open squares indicate the control, the open diamonds indicate 20
.mu.g/ml rBPI.sub.21, the open circles indicate 10 .mu.g/ml rBPI.sub.21,
the open triangles indicate 5 .mu.g/ml rBPI.sub.21, the diamonds with a
cross inside indicate 1 .mu.g/ml rBPI.sub.21, and the open circle with a
cross inside indicates 16 .mu.g/ml LBP. The results indicate a
dose-dependent inhibitory effect on cell growth by 7 hours, with the
decrease in turbidity at 20 .mu.g/ml rBPI.sub.21 suggesting that some cell
lysis has occurred.
Further experiments were performed to confirm this result and to examine
the effect of CaCl.sub.2 on growth inhibition of S. aureus L-phase
variants by BPI protein product. The L-phase variant cells were grown
overnight in HI broth supplemented with 3.5% NaCl, 10 mM CaCl.sub.2 and
1,000 units/ml penicillin G, and were inoculated into 96 well plates to
A.sub.600 =.about.0.02 in 100 .mu.l of the same broth supplemented with
2.5, 5 or 10 mM CaCl.sub.2. As a control, the BPI-susceptible
gram-negative organism E. coli J5 was inoculated from an overnight culture
into TYE broth (Difco, Detroit, Mich.). Two-fold serial dilutions of
rBPI.sub.21, diluted in 5 mm citrate and 150 mm NaCl to a total volume of
2.5 .mu.l, were added to each well to yield final rBPI.sub.21
concentrations of 50 to 0.77 .mu.g/ml. The cells were incubated at
37.degree. C. on a rotary shaker and cell growth as measured by was
followed. The results of these experiments are shown in FIGS. 11, 12 and
13 and confirm that BPI protein product inhibits growth of the L-phase
variant beginning at about 4 hours. The greatest inhibitory effect was
observed for the cells grown in broth supplemented with 2.5 mM CaCl. Under
these conditions, rBPI.sub.21 at concentrations as low as 3.12 .mu.g/ml
inhibited growth and caused a decrease in absorbance. Higher
concentrations of CaCl.sub.2 (5 and 10 mM) appeared to inhibit the action
of rBPI.sub.21 on the cells. The results of the control experiment with E.
coli J5 are shown in FIG. 14. By comparison the E. coli J5 cells were
immediately inhibited by rBPI.sub.21, but only at the three highest
concentrations (50, 25 and 12.5 .mu.g/ml).
EXAMPLE 4
IN VITRO EFFECTS OF BPI PROTEIN PRODUCTS ON STREPTOCOCCUS SPECIES IN A
RADIAL DIFFUSION ASSAY
Radial diffusion assays were conducted to evaluate the effect of the same
BPI protein products tested in Example 3, rBPI.sub.23, rBPI.sub.21,
rBPI.sub.50, BPI-Immunoglobulin fusion product and rBPI.sub.42 dimer, on
S. pneumoniae and S. pyogenes. L-phase variants of S. pneumoniae (ATCC
Accession No. 35088) were initially isolated by plating log-phase
bacterial forms on L-phase variant media (HI broth supplemented with 3.5%
NaCl, 10% horse serum, and 1000 units/ml penicillin G). The cells grew
optimally on agar plates consisting of 1.3% Bacto-agar (Difco, Detroit,
Mich.) and BPII broth supplemented with 2% NaCl and 10% inactivated horse
serum. The L-phase variants were subsequently adapted to grow on this BPII
medium lacking horse serum. Colonies of L-phase variants from the agar
plates were resuspended into BPII broth supplemented with 2% NaCl and
adjusted to A.sub.600 =.about.0.025. The cell suspension was added at a 1:
100 dilution to molten BPII agar medium containing 0.8% agarose and BHI
broth supplemented with 2% NaCl and 1000 units/ml penicillin G, which was
then allowed to solidify. Serial 2-fold dilutions of each BPI protein
product in unformulated titrate buffer or formulated titrate buffer were
added in 5 .mu.l aliquots to 3-mm wells prepared in the plates.
Unformulated citrate buffer was 20 mM citrate, pH 5.0, 150 mM NaCl for
rBPI.sub.23, or 5 mM titrate, pH 5.0, 150 mM NaCl for other BPI protein
products. Formulated titrate buffer was 20 mM citrate, pH 5.0, 150 mM
NaCl, with 0.1% poloxamer 188 and 0.002% polysorbate 80 for rBPI.sub.23,
or 5 mM titrate, pH 5.0, 150 mM NaCl with 0.2% poloxamer 188 and 0.002%
polysorbate 80 for rBPI.sub.21, or 5 mM titrate, pH 5.0, 150 mM NaCl with
0.1% poloxamer 188 and 0.002% polysorbate 80 for other BPI protein
products. The corresponding formulated or unformulated buffer was also
added to wells as a control. After 24 hours of incubation at 37.degree.
C., the plates were examined for zones of growth inhibition. There were no
differences in the activity of BPI protien products in formulated vs.
unformulated buffer.
The results of one representative assay, shown in FIG. 15, demonstrate that
the L-phase variant of S. pneumoniae is susceptible to all of these BPI
protein products, while the normal bacterial form is not (as shown above
in Example 3). In the figure, a closed square signifies rBPI.sub.21, an
open circle indicates rBPI.sub.23, an open triangle indicates rBPI.sub.50,
a closed inverted triangle indicates unglycosylated rBPI.sub.50, an open
square enclosing a plus-sign indicates BPI-Immunoglobulin fusion, and an
open square signifies rBPI.sub.42 dimer. These data show that rBPI.sub.50
was the most potent inhibitor of growth, followed by rBPI.sub.42 dimer and
BPI-Immunoglobulin fusion.
Similar results were obtained when the experiment was repeated with
penicillin-treated S. pneumoniae bacterial forms grown in the presence of
osmotic protection. Log-phase cells growing in BHI broth were concentrated
to A.sub.600 =.about.5.0, added to the same BHI agarose medium containing
penicillin as above, tested with serial dilutions of the same BPI protein
products as above, and incubated for 48-72 hours. These penicillin-treated
bacteria were also observed to be susceptible to all tested BPI protein
products.
The effects of BPI protein products on the bacterial form (ATCC No. 25663)
and L-phase variant (ATCC No. 27080) of S. pyogenes were evaluated in this
radial diffusion assay. The bacterial form was grown in BHI broth, and the
L-phase variant was grown first on ATCC medium No. 608 and later in medium
608 supplemented with penicillin G and 10% heat inactivated horse serum.
The cells were incorporated into molten BHI agarose (for the bacterial
forms), or molten medium 608 agarose supplemented with penicillin G and 1%
heat inactivated horse serum (for the L-phase variants), and various
concentrations of rBPI.sub.21, rBPI.sub.23, rBPI.sub.20 and rBPI.sub.42
dimer were tested. As previously observed for the other bacterial species
tested, none of the BPI protein products had an inhibitory effect at the
concentrations tested on the S. pyogenes bacterial form. The L-phase
variant was susceptible to all BPI protein products tested, rBPI.sub.21,
rBPI.sub.23, rBPI.sub.50, and rBPI.sub.42 dimer, with rBPI.sub.42 dimer
and rBPI.sub.23 being most potent and rBPI.sub.50 least (this lower
potency of rBPI.sub.50 was not seen with other bacterial species).
The effects of additional BPI protein products, BPI-derived peptides, on
the bacterial and L-phase variants of S. pneumoniae (ATCC No. 35088) and
S. pyogenes (bacterial form ATCC No. 25663; L-phase variant ATCC No.
27080) were also evaluated in this radial diffusion assay. The results,
depicted in FIGS. 16 and 17 (for S. pneumoniae L-phase variant and
bacterial form, respectively) and FIGS. 18 and 19 (for S. pyogenes L-phase
variant and bacterial form, respectively) demonstrate that, in general,
these peptides inhibited growth of the L-phase variant in a similar manner
to that observed with the S. aureus L-phase variant. Unlike S. aureus, the
S. pyogenes bacterial form was susceptible to XMP.2, XMP. 13, XMP.30 and
XMP. 102 as well as to XMP.48, while the S. pneumoniae bacterial form also
appeared to be slightly susceptible to high concentrations of XMP.2, XMP.
13, XMP.48 and XMP. 102, but not XMP.30. However, when the experiment was
repeated with XMP. 13 and XMP.30, the S. pneumoniae bacterial form
appeared to be susceptible to XMP.30. As previously observed, only the
L-phase variants were sensitive to rBPI.sub.42 dimer.
EXAMPLE 5
IN VITRO EFFECTS OF BPI PROTEIN PRODUCTS ON ENTEROCOCCUS FAECALIS L-PHASE
VARIANTS IN A RADIAL DIFFUSION ASSAY
Radial diffusion assays were conducted to evaluate the effect of the same
BPI protein products, used in Example 3, rBPI.sub.23, rBPI.sub.21,
rBPI.sub.50, BPI-Immunoglobulin fusion product and rBPI.sub.42 dimer, on
E. faecalis L-phase variants. L-phase variants of E. faecalis (ATCC
Accession No. 4200) were initially isolated by plating log-phase bacterial
forms on agar medium consisting of 1.3% Bacto-agar (Difco, Detroit, Mich.)
and BHI broth supplemented with 0.5% yeast extract, 0.93% NaCl, 9.73%
sucrose, 0.025% MgSO.sub.4, 10% heat inactivated horse serum and 1000
units/ml penicillin G (Medium 607, ATCC, Rockville, Md.), but also grew
well on BHI agar supplemented only with NaCl, sucrose and horse serum. The
cells were subsequently adapted to growth on Medium 607 lacking horse
serum. Colonies of L-phase variants from the agar plates were resuspended
into Medium 607 and adjusted to A.sub.600 =.about.0.025. The cell
suspension was added at a 1: 100 dilution to molten BHI agarose medium
containing BHI broth, 0.93% NaCl, 9.73% sucrose and 1000 units/ml
penicillin G, which was then allowed to solidify. Serial 2-fold dilutions
of each BPI protein product in 5 .mu.l were added to 3-mm wells prepared
in the plates. After 24 hours of incubation, the plates were examined for
zones of growth inhibition. The results of one representative assay, shown
in FIG. 20, demonstrate that the L-phase variant of E. faecalis is
susceptible to all of these BPI protein products, while the normal
bacterial form is not (as shown above in Example 3). In the figure, a
closed square signifies rBPI.sub.21, an open circle indicates rBPI.sub.23,
an open triangle indicates rBPI.sub.42 dimer, an open square enclosing a
plus-sign indicates BPI-Immunoglobulin fusion, and an open diamond
signifies rBPI.sub.50 Unglycosylated rBPIs0 behaved in an identical manner
to rBPI.sub.50. These data show that rBPI.sub.42 dimer and rBPI.sub.50
were the most potent inhibitors of growth.
Similar results were obtained when the experiment was repeated with
penicillin-treated E. faecalis bacterial forms grown in the presence of
osmotic protection. Log-phase cells growing in BHI broth were concentrated
to A.sub.600 =.about.5.0, added to the same BHI agarose medium (consisting
of medium 607 with penicillin G but without horse serum as in the radial
diffusion assay described above, tested with serial dilutions of
rBPI.sub.23, rBPI.sub.23, BPI-Immunoglobulin fusion and rBPI.sub.42 dimer,
and incubated for 48-72 hours. These penicillin-treated bacteria were also
observed to be susceptible to all tested BPI protein products. In this
experiment there were greater differences in activity, with rBPI.sub.42
dimer and BPI-Immunoglobulin fusion being the most potent inhibitors of
growth.
EXAMPLE 6
IN VITRO EFFECTS OF BPI PROTEIN PRODUCTS ON MYCOPLASMA IN RADIAL DIFFUSION
ASSAYS
Mycoplasmas are prokaryotes that lack a cell wall. Many members of this
group are naturally-occurring, non-pathogenic inhabitants of humans.
Mycoplasma pneumoniae, however, is a major cause of primary atypical
pneumonia. The susceptibility of L-phase variants to BPI protein products
predicts that mycoplasmas also are susceptible.
Experiments were performed to evaluate the effects of BPI protein products
on the mycoplasma Acholeplasma laidlawii, which is relatively unfastidious
and does not require a CO.sub.2 -enriched atmosphere to grow. Cells were
grown overnight in HI broth supplemented with 1% PPLO serum fraction
(Difco, Detroid, Mich.), incorporated into the same medium containing
agarose, and used in radial diffusion assays as described above for
L-phase variants. BPI protein products (rBPI.sub.23, BPI.sub.21,
BPI.sub.50, rBPI.sub.42 dimer and purified XMP.30) were added to the wells
and the plates were incubated at 37.degree. C. for 2 days. The results,
shown in FIG. 21, demonstrate that all of the BPI forms tested were
effective against the Acholeplasma. For rBPI.sub.23 and rBPI.sub.21, a
second zone of reduced growth was observed outside of the zone of complete
inhibition.
The experiment was repeated with the same BPI protein products and XMP.13,
XMP.2 and XMP.14. The results, depicted in FIG. 22, showed that XMP.13 and
XMP.30 formed inhibition zones while XMP.2 and XMP.14 were not active at
the concentrations tested. Additional experiments can be performed with
this Acholeplasma species as well as other mycoplasma species.
EXAMPLE 7
IN VITRO EFFECTS OF LBP DERIVATIVES ON L-PHASE VARIANTS OF S. AUREUS, S.
PNEUMONIAE AND E. FAECALIS IN A RADIAL DIFFUSION ASSAY
BPI protein products and LBP protein derivatives were evaluated in the
radial diffusion assays described above for their effect on the growth of
L-phase variants of various bacterial species, including S. aureus, S.
pneumoniae and E. faecalis. The compounds evaluated were rBPI.sub.50,
rBPI.sub.23, rBPI.sub.21, mature LBP protein (LBP.sub.50), LBP.sub.25,
LBP(1-197)/BPI(200-456) hybrid and BPI(1-199)/LBP(198-456) hybrid.
A plasmid encoding the LBP(1-197) BPI(200-456) hybrid was constructed by
combining appropriate portions of the two molecules via a ClaI restriction
site engineered into homologous locations in the DNA encoding the two
molecules. The first step necessary for the construction of the mammalian
expression vector pING4160 was the construction of two intermediate
plasmids to introduce a ClaI restriction site by overlap extension PCR
mutagenesis at the Ile-Asp at positions 197-198 in LBP (to generate
plasmid pML127) and the Ile-Asp at positions 199-200 in BPI(to generate
plasmid pML126). These were silent mutations which changed the nucleotide
sequence only and not the amino acid sequence. The next step was to
combine the amino terminal portion of LBP from pML127 with the carboxyl
terminal of BPI from pML126 at the homologous ClaI sites to generate the
intermediate plasmid pML128. The final step was then to subclone the
LBP-BPI insert from pML126 into a mammalian expression vector to generate
pING4160.
To construct plasmid pML127 (LBP with ClaI at 197-198), overlapping primers
were designed to incorporate the changes necessary to encode a ClaI
recognition site at the desired location. The template was pML125, a
plasmid containing an insert encoding full length LBP. The primers were
LBP-10, SEQ. ID. NO: 228, facing downstream, and LBP-11, SEQ. ID. NO: 229,
facing upstream. Two separate PCR reactions were carried out with primer
pairs LBP-Bsm, SEQ. ID. NO: 230, facing downstream, and LBP-11, to
generate a 600 bp fragment that was then digested with StuI and ClaI to
generate a 389 bp fragment, and primer pairs LBP-10 and LBP-8, SEQ. ID.
NO: 231, facing upstream, to generate a 328 bp fragment that was then
digested with ClaI and Bsu361 to generate a 148 bp fragment. The two
resulting fragments were then ligated to the Bsu36I-StuI vector fragment
from pML125 to generate the plasmid pML127.
To construct plasmid pML126 (BPI with ClaI at 199-200), overlapping primers
were designed to incorporate the changes necessary to encode a ClaI
recognition site at the desired location. The template was pML124, a
plasmid containing an insert encoding full length BPI protein. The primers
were BPI-63, SEQ. ID. NO: 232, facing downstream, and BPI-64, SEQ. ID. NO:
233, facing upstream. Two separate PCR reactions were carried out with
primer pairs BPI-40, SEQ. ID. NO: 234, facing downstream, and BPI-64, to
generate a 260 bp fragment that was then digested with PmlI and Clal to
generate a 170 bp fragment, and primer pairs BPI-7, SEQ. ID. NO: 235,
facing upstream, and BPI-63, to generate a 296 bp fragment that was then
digested with ClaI and BstXI to generate a 215 bp fragment. The two
resulting fragments were then ligated to the BstXI-PmlI vector fragment
from pML124 to generate the plasmid pML126.
To construct pML128, the intermediate plasmid encoding the
LBP(1-197)BPI(200-456) hybrid, the 620 bp HindllI-ClaI fragment encoding
the amino terminal region of BPI in the plasmid pML126 was replaced with
the corresponding HindllI-ClaI fragment from pML127 encoding the amino
terminal region of LBP. To construct the mammalian expression vector
pING4160, the 623 bp FspI-Bsu36I fragment of pML128 was ligated to the 361
bp SalI-FspI fragment from pING4539 (described in Gazzano-Santaro et al.,
U.S. Application Ser. No. 08/261,660 filed Jun. 17, 1994), which includes
the LBP signal sequence, and the approximately 8630 bp Bsu36I-SalI
fragment from pING4321. The latter fragment includes sequences encoding
part of the carboxyl terminus of BPI and all the vector sequences, which
include the CMV promoter and the light chain 3' transcription termination
sequences (as described in Ammons et al., U.S. Application Ser. No.
08/212,132, filed Mar. 11, 1994).
A plasmid encoding the BPI(1-199) LBP(198-456) hybrid was constructed by
combining appropriate portions of the two molecules via a ClaI restriction
site engineered into homologous locations in the DNA encoding the two
molecules. The intermediate plasmid encoding the BPI(1-199)LBP(198-456)
hybrid, pML129, was constructed by replacing the 620 bp HindIII-Clal
fragment encoding the amino terminal region of LBP in the plasmid pML127
with the corresponding HindIII-Clal fragment from pML126 encoding the
amino terminal region of BPI protein.
To construct the mammalian expression vector pING4161, the 881 bp
BstBI-SstII/T4 fragment of pML129, including part of the BPI and all of
the LBP insert sequence, was ligated to the approximately 8755
XhoI/T4-BstBI fragment of pING4147 (described in Gazzano-Santaro et al.,
U.S. Application Ser. No. 08/261,660 filed Jun. 17, 1994). The latter
fragment includes sequences encoding the signal sequence and part of the
amino terminus of BPI and all the vector sequences, which include the CMV
promoter and the light chain 3' transcription termination sequences.
To obtain the desired hybrid proteins, beads co-cultured with CHO-K1 cells
transfected with pING4160 or pING4161 as described in U.S. patent Appl.
Ser. No. 08/072,063 filed May 19, 1993, were washed with approximately 600
mls of 20 mM sodium acetate, pH 4.0 mM NaCl and then 600 mls of the same
buffer containing 600 mM NaCl. Protein was eluted in two steps of 20 mM
sodium acetate; the first with 1.0 M NaCl and the second with 1.5 M NaCl,
with the majority of the desired protein eluting from the S-Sepharose in
the 1.0M step. Fractions containing the protein were then pooled and
diluted to a final NaCl concentration of 300 mM with the addition of MES
buffer, to a final concentration of 20 mM ME-S, pH 5.0. The diluted
material recovered from all cell harvests was combined, yielding a final
volume of approximately 6.5 liters. This pooled eluate was applied to two
columns arranged in a tandem fashion, the first being a 100 ml Q-Sepharose
column and the second a 12 ml CM-Spherodex column. The flow through
material, which contained the desired protein, was adjusted to pH 4.0 and
loaded in three batches on to a 15 ml S-Sepharose column. Each time the
column was washed with 20 mM MES, pH 4.0 200 mM NaCl and the bound protein
recovered with a step elution of 20 mM MES, pH 5.5, 1.2M NaCl. The volume
of the recovered protein was approximately 40 mls. This material was then
run on a S-100 size exclusion column in 5 mM sodium citrate, pH 5.0, 150
mM NaCl. Column fractions were assayed using Coommassie stained SDS-PAGE
and Western analysis using an anti-LBP primary antibody, and fractions
containing the desired protein were pooled.
Radial diffusion assays on L-phase variants of S. pneumoniae (ATCC
Accession No. 35088) and E. faecalis (ATCC Accession No. 4200) and the
natural L-variant of S. aureus (ATCC Accession No. 19640) were performed
as described above in Examples 3, 5 and 6, using varying concentrations of
rBPI.sub.50, LBP.sub.50, LBP-BPI hybrid, and thaumatin as a control.
LBP-BPI hybrid inhibited growth of S. aureus and S. pneumoniae L-phase
variants, but not growth of E. faecalis L-phase variant. The LBP-BPI
hybrid was especially potent against S. pneumoniae, with activity close to
that of rBPI.sub.50. Results of one representative assay on S. aureus
L-phase variant using rBPI.sub.23, rBPI.sub.50, rBPI.sub.21, rLBP.sub.25,
rLBP.sub.50, LBP-BPI hybrid and BPI-LBP hybrid are displayed in FIG. 23.
The LBP-BPI hybrid was bactericidal while rLBP.sub.50 and rLBP.sub.25 had
little or no activity. The BPI-LBP hybrid was also active against the
L-phase variant but less so than the LBP-BPI hybrid. As usual, rBPI.sub.42
dimer was the most potent molecule against the L-phase variant. These
results suggest that the presence of the BPI carboxyl terminus has
enhanced the activity of rLBP.sub.25 on prokaryotic membranes.
Further radial diffusion assays on these three organisms were conducted
with LBP derivatives, LBP peptide 1-2, SEQ. ID NO: 236, and LBP peptide
2-1, SEQ. ID NO: 237, and with other BPI protein products, rBPI.sub.42
dimer, XMP.2, XMP.13, XMP.14, XMP.3 and XMP.5. Results of representative
assays on the S. aureus bacterial form and L-phase variant are displayed
in FIGS. 24 and 25. At high concentrations, LBP peptide 1-2 had some
bactericidal activity against the bacterial form. For the L-phase variant,
LBP peptide 2-1 (amino acids 73-99 from domain II of LBP) was as potent as
XMP.3 (amino acids 73-99 from domain II of BPI protein). XMP.5, XMP.13 and
rBPI.sub.42 dimer also displayed bactericidal activity against the L-phase
variant, with rBPI.sub.42 dimer being the most potent of all molecules
tested. Additional assays were performed to evaluate the activity of LBP
peptide 1-2 and LBP peptide 2-1 and other BPI protein products on the E.
faecalis bacterial form, the S. pyogenes L-phase variant of Example 4, and
the S. pneumoniae bacterial form and L-phase variant. LBP peptide 1-2
displayed some bactericidal activity against the two bacterial forms at
high concentrations (300 pmol/well or more). LBP peptide 1-2 was as potent
as rBPI.sub.42 dimer against the S. pneumoniae L-phase variant, and was
intermediate in potency between rBPI.sub.42 dimer and XMP.13 for the S.
pyogenes L-phase variant. LBP peptide 2-1 had no bactericidal activity
against the tested bacterial forms or L-phase variants.
EXAMPLE 8
IN VITRO EFFECTS OF BPI-DERIVED PEPTIDES ON S. AUREUS IN A RADIAL DIFFUSION
ASSAY
This example addresses in vitro screening of BPI protein products,
specifically BPI-derived peptides, for antimicrobial activity in a radial
diffusion assay. The peptides tested were all prepared according to the
procedures described in parent U.S. patent application Ser. Nos.
08/209,762 and 08/183,222. Briefly summarized, peptides were prepared by
solid phase peptide synthesis according to the methods of Merrifield, J.
Am Chem. Soc. 85: 2149 (1963) and Merrifield et al. Anal. Chem., 38:
1905-1914 (1966) using an Applied Biosystems, Inc. Model 432 peptide
synthesizer. Peptide design was based in principal part on the discovery
of three functional domains present in the NH.sub.2 -terminal region of
the BPI holoprotein domain I comprising BPI amino acids from about
position 17 to about position 45 (SEQ ID NO: 1); domain II comprising BPI
amino acids from about position 65 to about 99 (SEQ ID NO: 6); and domain
III comprising BPI amino acids from about position 142 to about position
169 (SEQ ID NO: 12). Peptides included sequences and subsequences of the
domain sequences and variants thereof including linear and branched chain
combination peptides with and without single or multiple amino acid
(including atypical amino acid) substitutions as well as cyclized peptides
and interdomain sequence peptides. Table 1 below sets out peptides derived
from or based on BPI sequences, as identified by the peptide number with a
prefix XMP or BPI (e.g., XMP.1 or BPI.1, XMP.2 or BPI.2, etc.), SEQ ID NO:
, amino acid sequence based on reference to position within BPI and
designation of amino acid substitutions and additions. Also set out in
Table 1 are mass spectroscopy and HPLC estimates of purity of the
peptides.
Overnight S. aureus cultures were diluted 1: 50 into fresh tryptic soy
broth and incubated for 3 hours at 37.degree. C. to attain log phase.
Bacteria were pelleted at 3,000 rpm (1500 x g) for 5 minutes in a Beckman
J-6M centrifuge. 5 ml of 10 mM sodium phosphate buffer, pH 7.4, was added
and the suspension was recentrifuged. Supernatant was decanted and 5 ml of
fresh buffer was added for an OD.sub.570 determination. An OD.sub.570 of
1.0 was considered equivalent to 5.times.10.sup.8 CFU/ml. Ten mL of molten
underlayer agarose comprising 3% tryptic soy broth, 1% agarose (Pharmacia,
Piscataway, N.J.), 0.02% Tween 20, and 10 mM sodium phosphate, at pH 7.4
was added to polystyrene tubes and maintained in a 56.degree. C. water
bath until the addition of bacteria. Tubes were cooled to approximately
45.degree. C., bacteria were added to give a final concentration of
1.times.10.sup.6 CFU/ml, and the tubes were mixed again by inverting. The
contents were poured into level square petri dishes and distributed
evenly. The agarose solidified in less than 30 seconds and had a uniform
thickness of about 1 mm. A series of wells were punched into the hardened
agarose using a sterile 3 mm punch attached to a vacuum apparatus.
Peptides to be screened were 2-fold serially diluted in Dulbecco's PBS
(D-PBS) starting from a concentration of approximately 1 mg/mL. Five .mu.L
of each dilution was added to each well and the plates were incubated at
37.degree. C. for 3 hours. An overlayer of 10 mL of molten agarose
comprising 6% tryptic soy broth, 1% agarose, and 10 mM sodium phosphate,
pH 7.4, (at approximately 45.degree. C.) was then added and plates were
incubated overnight at 37.degree. C. Following this overnight incubation,
a dilute Coomassie solution was poured into the plates and allowed to
stain for 24 hours.
Clear zones of growth inhibition around each well were measured with
calipers. The actual area of growth inhibition (mm.sup.2) was calculated
by subtracting the area of the well. Table 1 below sets out the results of
the radial diffusion assays for tested peptides in terms of the number of
.mu.g or picomoles (pmol) of peptide required to establish a 30 mm.sup.2
area of growth inhibition.
Peptides XMP.221 through XMP.281 (SEQ ID NOs: 166 through 26) are prepared
and tested for anti-bacterial activity as described above.
TABLE 1
__________________________________________________________________________
S. aureus
Peptide # MS % HPLC %
.mu.g/ pmol/
(Seq. ID No.)
Protein AA Segment Purity
Purity
30 mm.sup.2 zone
30 mm.sup.2
__________________________________________________________________________
zone
XMP.1 (4)
19-33 -- 2 Peaks
N N
XMP.2 (7)
85-99 64 37.2 N N
XMP.3 (11)
73-99 -- 17 X X
XMP.4 (3)
25-46 -- No Peak
N N
XMP.5 (67)
142-163 -- 18 X X
XMP.7 (54)
(90-99) .times. 2 69 27 >5.00 >1,891
XMP.8 (8)
90-99 79 Mixtures
X X
XMP.9 (51)
95-99,90-99 -- 29 X X
XMP.10 (55, 65)
94-99, 90-99, 90-99 and
-- Mixture
X X
95-99, 90-99, 90-99
XMP.11 (13)
148-151,153-161 -- 76 X X
XMP.12 (14)
141-169 -- 26 X X
XMP.13 (15)
148-161 78 69 >5.00 >2,924
XMP.13P (15)
148-161 100 98 X X
XMP.14 (2)
21-50 -- -- X X
XMP.15 (16)
85-99, A @ 85 (I) 66 57.6 X X
XMP.16 (17)
85-99, A @ 86 (K) -- 84.1 X X
XMP.17 (18)
85-99, A @ 87 (I) 86 77,67 X X
XMP.18 (19)
85-99, A @ 88 (S) 66 70 X X
XMP.19 (20)
85-99, A @ 89 (G) -- 69 X X
XMP.20 (21)
85-99, A @ 90 (K) -- 66 X X
XMP.21 (22)
85-99, A @ 91 (W) 68 66.8 X X
XMP.22 (23)
85-99, A @ 92 (K) -- 66 X X
XMP.23 (24)
85-99, A @ 94 (Q) -- 69 X X
XMP.24 (25)
85-99, A @ 95 (K) -- 67 X X
XMP.25 (26)
85-99, A @ 96 (R) -- 73 X X
XMP.26 (27)
85-99, A @ 97 (F) -- 73 X X
XMP.27 (28)
85-99, A @ 98 (L) -- 65 X X
XMP.28 (29)
85-99, A @ 99 (K) -- 80 X X
XMP.29 (56)
(148-161) .times. 2
-- 26 >5.00 >1,469
XMP.30 (52)
90-99,148-161 -- 21 3.68 1,216
XMP.30-P (52)
90-99,148-161 95 98 X X
XMP.31 (33)
148-161, A @ 148 (K)
-- 68 X X
XMP.32 (34)
148-161, A @ 149 (S)
-- 70 X X
XMP.33 (35)
148-161, A @ 150 (K)
-- 58 X X
XMP.34 (36)
148-161, A @ 151 (V)
-- 51 X X
XMP.35 (37)
148-161, A @ 152 (G)
-- 72 X X
XMP.36 (38)
148-161, A @ 153 (W)
-- 64 X X
XMP.37 (39)
148-161, A @ 154 (L)
-- 51 X X
XMP.38 (40)
148-161, A @ 155 (1)
-- 70 X X
XMP.39 (41)
148-161, A @ 156 (Q)
-- 53 X X
XMP.40 (42)
148-161, A @ 157 (L)
-- 53 X X
XMP.41 (43)
148-161, A @ 158 (F)
-- 63 X X
XMP.42 (44)
148-161, A @ 159 (H)
-- 59 X X
XMP.43 (45)
148-161, A @ 160 (K)
-- 53 X X
XMP.44 (46)
148-161, A @ 161 (K)
-- 70 X X
XMP.45 (31)
85-99, A @ 94(Q) & 95(K)
71 46 >5.00 >1,697
XMP.46 (57)
(90-99) .times. 2, A @ 1st 94(Q) & 95(K)
67 47 >5.00 >1,811
XMP.47 (58)
(90-99) .times. 2, A @ 2d 94(Q) & 95(K)
57 34 >5.00 >2,461
XMP.48 (59)
(90-99) .times. 2, A @ both 94(Q) & 95(K)
68 33 >5.00 >1,390
XMP.54 (5)
21-35 -- -- X X
XMP.55 (61)
152-172 -- 28 >5.00 >1,592
XMP.56 (47)
85-99, K @ 94 (Q) &
-- 55 N N
Q @ 95(K)
XMP.57 (99)
Cys 85-99 Cys 50 Mixture
N N
XMP.58 (9)
Cys-85-99 49 25.7 N N
XMP.59 (30)
85-99, A @ 90(K) & 92(K)
56 30 N N
XMP.60 (32)
85-99, A @ 86(K) & 99(K)
57 78 N N
XMP.61 (48)
85-99, F @ 91(W) 60 60 N N
XMP.63 (53)
85-99, 148-161 38 31.0 >5.00 >1,006
XMP.65 Rd (68)
Cys-85-99-Cys 41 22, 34
N N
XMP.65 Ox (10)
Cys-85-99-Cys -- No Peak
>5.00 >3,118
XMP.66 (49)
85-99, W.sub.D @ 91(W)
-- 70 N N
XMP.67 (50)
85-99, .beta.-(1-naphthyl)-A
65 52 N N
@ 91
XMP.69 (60)
[90-99, A @ 94 (Q) &
44 54,40 3.83 1,058
95 (K)] .times. 3
XMP.70 (63)
85-99, .beta.-(3-pyridyl)-A
66 54 N N
@ 91
XMP.71 (64)
A.sub.D -A.sub.D -85-99
-- 60 N N
XMP.72 (66)
85-99, .beta.-(3-pyridyl)-A
-- 52 N N
@ 97 (F)
XMP.73 (62)
85-99, F @ 95 (K) -- 44, 39
>5.00 >1,811
XMP.74 (70)
148-161, 90-99 -- 29 >5.00 >2,148
XMP.75 (100)
IKKRAISFLGKKWQK (2-mixed)
-- 32 >5.00 >2,031
XMP.76 (71)
85-99, F, @ 95 (K) 53 39 N N
XMP.77 (72)
85-99, W @ 95 (K) -- 38 3.15 1,684
XMP.79 (73)
85-99, K @ 94 (Q) -- 48 N N
XMP.80 (74)
85-99, .beta.-(1-naphthyl)-A
71 44 4.82 2,533
@ 95 (K)
XMP.81 (75)
85-99, F @ 94 (Q) 44 33, 35
>5.00 >2,345
XMP.82 (76)
148-161, W @ 158 (F)
82 58 >5.00 >1,198
XMP.83 (77)
148-161, .beta.(1-naphthyl)-A
85 63 >5.00 >2,034
@ 153 (W)
XMP.84 (78)
85-99, .beta.-(1-naphthyl) A @
64 50 >5.00 >2,017
91 (W) & F @ 95 (K)
XMP.85 (79)
148-161, L @ 152 (G)
79 74 >5.00 >1,881
XMP.86 (80)
148-161, L @ 156 (Q)
69 51 >5.00 >2,048
XMP.87 (81)
148-161, L @ 159 (H)
79 63 >5.00 >1,536
XMP.88 (82)
85-99, F @ 94 (Q) 62 50 >5.00 >2,380
& 95 (K)
XMP.89 (84)
85-99, .beta.-(1-naphthyl) A @
66 50 >5.00 >1,882
91 (W) & F @ 94 (Q)
XMP.90 (85)
85-99, .beta.-(1-naphthyl) A @ 91 (W),
70 63 >5.00 >1,863
F @ 94 (Q) & 95 (K)
XMP.91 (86)
148-161, F @ 156 -- 31 N N
(Q)
XMP.92 (87)
148-161, K @ 156 (Q)
-- 50 N N
XMP.93 (88)
85-99 148-161 .beta.-(1-naphthyl) A
72 38 >5.00 >980
@ 91 (W), F @ 95 (K)
XMP.94 (89)
148-161, F @ 159 -- 59 >5.00 >922
XMP.95 (90)
148-161, F @ 152 (G)
-- 57 N N
XMP.96 (101)
148-161, F @ 161 (K)
-- 60 >5.00 >2,048
XMP.97 (92)
148-161, K @ 152 (G)
-- 67 N N
XMP.98 (83)
90-99, .beta.-(1-naphthyl) A @ 91 (W),
69 31 N N
F @ 95 (K) + 148-161 F @ 156 (Q)
XMP.99 (93)
[90-99, W @ 95 -- -- >5.00 >1,064
(K)] .times. 3
XMP.100 (94)
148-161, K @ 152 (G) &
-- 61 N N
156 (Q)
XMP.101 (95)
(148-161) .times. 2[K @ 152(G) & 156(Q),
-- 16 >5.00 >993
F @ 159(H) & 161(K)]
XMP.102 (96)
90-99 (F @ 95(K)) + 148-161 L @ 156
-- 16 N N
(Q)
XMP.103 (102)
85-99, W @ 94 (Q) -- 28 >5.00 >2,703
XMP.104 (103)
148-161, S @ 156 (Q)
-- 34 >5.00 >5,569
XMP.105 (104)
85-99, .beta.-(1-naphthyl)-A @ 94 (Q)
58 43 >5.00 >1,843
XMP.106 (105)
148-161, T @ 156 (Q)
-- 26 N N
XMP.107 (106)
148-161, W @ 159 (H)
-- 55 N N
XMP.108 (107)
148-161, W @ 161 (K)
-- 50 >5.00 >3,219
XMP.109 (108)
148-161, .beta.(1-naphthyl)-A
-- 41 N N
@ 158 (F)
XMP.110 (109)
148-161, .beta.(1-naphthyl)-A
-- 56 N N
@ 159 (H)
XMP.111 (110)
148-161, .beta.(1-naphthyl)-A
-- 73 >5.00 >2,809
@ 161 (K)
XMP.112 (111)
85-99, .beta.(1-naphthyl)A
-- 56 N N
& 91 (W) & 95 (K)
XMP.113 (112)
148-161, F @ 157 (L)
-- 46 N N
XMP.114 (113)
KWQLRSKGKIKIFKA -- 17 N N
XMP.116 (114)
148-161, K @ 152 (G), .beta.(1-naphthyl)A
-- 72 N N
@ 153 (W)
XMP.119 (115)
85-99, .beta.(1-naphthyl)A @ 91 (W)
-- 77 >5.00 >2,617
& 94 (K)
XMP.120 (116)
85-99, K @ 97 (F) -- 52 N N
XMP.121 (117)
85-99, .beta.(1-naphthyl)A @ 94 (Q)
65 35 >5.00 >2,540
& 95 (K)
XMP.122 (118)
85-99, .beta.(1-naphthyl)A @ 91 (W),
-- 46 >5.00 >2,526
94(Q) & 95 (K)
XMP.123 (119)
148-161, p-Amino-F @ 156 (Q)
-- 64 N N
XMP.124 (120)
148-161, K @ 152(G), W @
-- 67 N N
158 (F)
XMP.125 (121)
148-161, Y @ 156 (Q)
-- 54 N N
XMP.126 (122)
148-161, W.sub.D @ 153 (W)
66 54 N N
XMP.127 (123)
148-161, F @ 153 (W)
65 63 N N
XMP.128 (124)
148-161 F.sub.D @ 153 (W)
63 51 N N
XMP.129 (125)
148-161, 1-.beta.(1-naphthyl)A.sub.D @
24 28 N N
153 (W)
XMP.130 (126)
148-161, 2-.beta.(1-naphthyl)A @
55 80 N N
153 (W)
XMT.131 (127)
148-161, 2-.beta.(1-naphthyl)A.sub.D @
75 60 N N
153 (W)
XMP.132 (128)
148-161, Pyr-A @ 153 (W)
49 50 N N
XMP.133 (129)
148-161, p-Amino-F @ 153 (W)
63 47 N N
XMP.134 (130)
148-161, p-Amino-F @ 152 (G)
-- 68 N N
XMP.135 (131)
148-161, K @ 153 (W)
-- 70 N N
XMP.136 (132)
85-99, E @ 95 (K) -- 50 N N
XMP.137 (133)
Cys-148-161-Cys -- 28 X X
XMP.138 (134)
148-161, K @ 152 (G), F @ 153 (W)
-- 61 N N
XMP.139 (135)
148-161, Y @ 153 (W)
-- 60 N N
XMP.140 (136)
90-99 .beta.(1-naphthyl)A @ 94 (Q)
-- 26 >5.00 >1,601
& 95 (K) + 104
XMP.141 (137)
85-99, W @ 97 (F) -- 50 N N
XMP.142 (138)
148-161, W @ 157 (L)
-- 57 N N
XMP.143 (139)
148-161, .beta.(1-naphthy)A
-- 65 N N
@ 157 (L)
XMP.144 (140)
148-161, Cyclohexyl-A
-- 60 N N
@ 153 (W)
XMP.145 (141)
90-99, .beta.(1-naphthyl)A @ 94(Q)
-- 20 X X
& 95(K) + 148-161
148-161, .beta.(1-naphthyl)A @
-- 53 N N
XMP.146 (142)
59(H) & 161(K)
XMP.147 (143)
85-99 K @ 96 (R) -- 55 N N
XMP.148 (144)
148-161, .beta.(1-naphthyl)A
-- 62 N N
@ 153 (W) & 159 (H)
XMP.149 (147)
KWKVFKKIEK + 148-161
-- 27 N N
XMP.150 (148)
KWAFAKKQKKRLKRQWLKKF
-- Mixture
N N
XMP.151 (55)
94-99, 90-99, 90-99
-- 14 N N
XMP.152 (65)
95-99, 90-99, 90-99
-- 21 N N
XMP.153 (149)
(90-99) .times. 3 -- 17 N N
XMP.154 (150)
(90-99) .times. 2, .beta.(1-naphthyl) A
-- 31 >5.00 >1,796
@ 1st 94 (Q) & 95 (K)
XMP.155 (151)
(90-99) .times. 2, .beta.(1-naphthyl) A
-- 23 >5.00 >1,796
@ 2nd 94 (Q) & 94 (K)
XMP.156 (152)
(90-99) .times. 2, .beta.(1-naphthyl) A
-- 38 N N
@ both 94 (Q) & 95 (K)
XMP.157 (153)
(90-99, .beta.(1-naphthyl) A
-- 38 N N
@ 94 (Q) & 95 (K)) .times. 3
OXMP.158 (154)
85-99, 148-161, .beta.(1-naphthyl) A
-- 16 N N
@ 94 (Q) & 95 (K)
XMP.159 (155)
(90-99, .beta.(1-naphthyl) A
-- 23 >5.00 >1,590
@ 91 (W) & 95 (K)) + 82
XMP.160 (156)
(90-99) .times. 2, .beta.(1-naphthyl) A @
-- 32 >5.00 >1,782
both 91. (W) & 95 (K)
XMP.161 (157)
148-161, K @ 152 (G) & A @ 153 (W)
-- 75 >5.00 >2,999
XMP.162 (158)
90-99, 148-161, W @ 95 (K)
-- 21 N N
XMP.163 (159)
(90-99) .times. 2, W @ both 95 (K)
-- Mixture
>5.00 >1,810
XMP.164 (160)
(90-99) .times. 2, .beta.(1-naphthyl) A @ both
-- 46 >5.00 >1,796
94 (Q)
XMP.165 (161)
(90-99, .beta.(1-naphthyl A @ 91 (W)
-- 72 >5.00 >1,847
& F @ 95 (K)) .times. 2
XMP.166 (162)
148-161, V @ 153 (W)
-- 68 N N
XMP.167 (163)
90-97 -- 56 N N
XMP.168 (164)
C- 90-101-C -- 13 N N
XMP.169 (165)
C-90-97-C -- 20 >5.00 >4,974
XMP.170 (227)
90-101 -- 60 N N
__________________________________________________________________________
X = Not tested
N = No detectable activity up to 5 .mu.g/well
EXAMPLE 9
IN VITRO EFFECTS OF A VARIETY OF BPI PROTEIN PRODUCTS ALONE OR IN
COMBINATION WITH ANTIBIOTICS ON STRAINS OF STREPTOCOCCUS PNEUMONIAE
This experiment evaluated the in vitro growth inhibitory effects of a
variety of BPI protein products, alone or in combination with antibiotics,
on clinical isolates of Streptococcus pneumoniae (from Baxter
Microscan.RTM. library, Sacramento, Calif.). The BPI protein products'
direct growth inhibitory effect and effect on the antibiotic
susceptibility of the organisms was determined using Microscan.RTM. panel
plates (Baxter Diagnostics, Inc., Deerfield, Ill.) that allow simultaneous
determination of minimum inhibitory concentrations for a number of
different antibiotics. Any other antimicrobial panel systems known in the
art, such as the Pasco (DIFCO, Detroit, Mich.) and Alamar (Alamar,
Sacramento, Calif.) systems, may be used instead of the Microscan.RTM.
system to assay for activity. Control assays performed with the
Microscan.RTM. panel plates confirmed that the formulation buffer for
rBPI.sub.21 (without rBPI.sub.21) had no effect on the antibiotic
susceptibility of a variety of organisms: S. pneumoniae (Microscan library
no. 31573), S. pyogenes (Group A) (30403), S. bovis (008-010), S. aureus
(052-106) and E. faecalis (011-066).
The antimicrobial susceptibility tests performed on the Microscan.RTM.
panel plates are miniaturizations of the broth dilution susceptibility
test. Antimicrobial agents are serially diluted in Mueller-Hinton broth
(supplemented with calcium and magnesium, or with sodium chloride for
oxacillin, or with thymidine phosphorylase for trimethoprim,
sulfamethoxazole and trimethoprim/sulfamethoxazole) to concentrations
bridging the range of clinical interest. One well on the 96-well
Microscan.RTM. plate is a growth control well that contains dehydrated
broth only. The remaining wells contain dehydrated broth and antibiotic
(or broth and biochemical reagent indicator), which is rehydrated to the
desired concentration by inoculation of a standardized suspension of test
organism. The chromogenic biochemical agent indicators are used to
identify and characterize the species of bacteria based on detection of pH
changes and substrate utilization. After incubation overnight, the minimum
inhibitory concentration (MIC) of an antibiotic for the test organism is
determined by observing the well with the lowest concentration of the
antibiotic that shows inhibition of growth. Two types of panel plates were
utilized to test these gram-positive organisms: the Pos Combo Panel Type
6, and the Pos MIC Panel Type 6 (both available from Baxter Diagnostics,
Inc., Deerfield, Ill.). The concentrations of antibiotics tested in each
panel are shown in Tables 2 and 3 below.
TABLE 2
__________________________________________________________________________
Antibiotic Concentrations (.mu.g/mL) Tested in Pos Combo Panel Type 6
Plate
__________________________________________________________________________
Trimethoprim/Sulfamethoxazole
2/38
1
Rifampin 2 1
Imipenem 8 4
Cephalothin 16 8
Amoxicillin/K Clavulanate
16/8
8/4
4/2
Cefotaxime 32 8
Ciprofloxacin 2 1
Norfloxacin 8 4
Nitrofurantoin 64 32
Gentamicin 6 4 2 1
Clindamycin 2 1 0.5
0.25
Cefazolin 16 8 4 2
Erythomycin 4 2 1 0.5
0.25
Vancomycin 16 8 4 2
Penicillin 8 4 2 1 0.5
0.25
0.12
0.06
0.03
Ampicillin 8 4 2 1 0.5
0.25
0.12
Oxacillin 4 2 1 0.5
Tetracycline 128
8 4 2
__________________________________________________________________________
TABLE 3
__________________________________________________________________________
Antibiotic Concentrations (.mu.g/mL) Tested in Pos MIC Panel Type 6
__________________________________________________________________________
Plate
Erythromycin 4 2 1 0.5
0.25
Vancomycin 16 8 4 2
Clindamycin 2 1 0.5
0.25
Cefazolin 16 8 4 2
Cephalothin 16 8 4 2
Cefuroxime 16 8 4 2
Ceftriaxone 32 16 8 4
Chloramphenicol 16 8 4
Cefotaxime 32 16 8 4
Ticarcillin/K Clavulanate
8 4 2 1
Imipenem 8 4 2 1
Gentamicin 6 4 2 1
Amoxicillin/K Clavulante
16/8
8/4
4/2
Ampicillin/Sulbactam
16/8
8/4
Rifampin 2 1
Trimethoprim/Sulfamethoxazole
2/38
Norfloxacin 8 4
Penicillin 8 4 2 1 0.5
0.25
0.12
0.06
0.03
Ampicillin 8 4 2 1 0.5
0.25
0.12
Oxacillin 4 2 1 0.5
Tetracycline 128
8 4 2
Nitrofurantoin 64 32
Ciprofloxacin 2 1
Amikacin 32 16
Sulfamethoxazole
256
__________________________________________________________________________
For each experimental run, the following procedure was performed: The
organism was streaked onto 5% sheep blood agar plates (Remel, Lenexa,
Kans.) and incubated for 18-24 hours overnight. Well-isolated colonies
from the plates were emulsified in 3 ml of sterile Inoculum Water (catalog
no. B1015-2, MicroScan.RTM. system, Baxter Diagnostics, Inc., Deerfield,
Ill.) to a final turbidity equivalent to 0.5 McFarland Barium Sulfate
standard. This cell suspension was vortexed for 2 to 3 seconds and 100
.mu.l was transferred to glass tubes containing 25 ml of Inoculum Water
with Pluronic-D (catalog no. B1015-7, MicroScan.RTM. system, Baxter
Diagnostics, Inc., Deerfield, Ill.) (hereinafter "Pluronic Inoculum
Water"), or 25 ml of Pluronic Inoculum Water into which the BPI protein
product (in formulation buffer) had been diluted to the desired
concentration, generally between 0 and 64 .mu.g/mL.
The 25 ml of this inoculum containing the BPI protein product was mixed by
inversion and poured into a tray. The inoculum was drawn up into a manual
96-well pipetting system (RENOK.TM. rehydrator-inoculator system, Baxter
Health Care Corporation, West Sacramento, Calif.) designed for use with
the Microscan.RTM. panel plates, and 110 .mu.l of the inoculum was
delivered to each well of a Microscan.RTM. Pos Combo Panel Type 6 or Pos
MIC Panel Type 6 plate. When added to the wells, this inoculum achieves a
final bacterial concentration of 4.times.10.sup.5 to 7.times.10.sup.5
CFU/ml. The panel plates were then incubated at 35.degree. C. for 15-24
hours and read visually for cell growth.
No growth was defined as a slight whiteness in the well or a clear broth.
Growth appeared as turbidity which could take the form of a white haze
throughout the well, a white button in the center of the well, or a fine
granule growth throughout the well. All wells were read against a black
indirectly lighted background. Visual results of the biochemical reactions
were read into a database for bacterial identification. The MICs for each
antibiotic tested were determined by identifying the lowest concentration
of antibiotic which inhibited visible growth.
Table 4 below displays a summary of the results of the antibiotic screening
panels, reported for each strain tested as the MIC of the tested
antibiotics at varying concentrations of rBPI.sub.21. Results are only
reported where rBPI.sub.21 altered the antibiotic susceptibility. The
antibiotic susceptibility standards (interpretation of an MIC as
clinically resistant, intermediate or susceptible according to NCCLS
standards) applicable to the organism tested appear in Table 4A. Stars
after the antibiotic name in the "antibiotic tested" column indicate
whether rBPI.sub.21 reversed the resistance of that organism to the
antibiotic tested (two stars) or converted an indifferent MIC into a
susceptible MIC (one star). These data show that rBPI.sub.21 converted one
ampicillin-resistant strain of S. pneumoniae into ampicillin-susceptible
(the other tested strains were already sensitive to ampicillin) and
increased the susceptibility of other S. pneumoniae strains to amikacin,
ampicillin, gentamicin and penicillin.
Table 4 also shows the presence or absence of bacterial growth in the
growth control wells, which contained varying concentrations of
rBPI.sub.21 alone without antibiotic. A "G" signifies growth at the tested
concentration, while "NG" signifies no growth. These results indicate that
S. pneumoniae is a BPI-susceptible organism; rBPI.sub.21 was directly
bactericidal/growth inhibitory for all tested isolates of S. pneumoniae at
a concentration of 2 .mu.g/ml.
Additional screening of S. pneumoniae strains was conducted with a variety
of BPI protein products, rBPI.sub.21, rBPI.sub.23, rBPI.sub.50 and
rBPI.sub.42 dimer. Table 4X below displays a summary of the results of the
antibiotic screening panels, reported for each strain tested as the MIC of
the tested antibiotics at varying concentrations of the indicated BPI
protein product. Results are only reported if antibiotic susceptibility
was altered.
TABLE 4
______________________________________
EFFECTS OF rBPI.sub.21 .+-. ANTIBIOTICS ON
Streptococcus pneumoniae
Minimum Inhibitory Concentration of
Antibiotic (.mu.g/mL)
Micro- With With With With With
scan 0 0.5 2 8 16
Library
Antibiotic
.mu.g/mL
.mu.g/mL
.mu.g/mL
.mu.g/mL
.mu.g/mL
ID No. Tested rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
______________________________________
31573 -- G G NG NG NG
Ampici- 8 <0.12 <0.12 <0.12 <0.12
llin**
Penicillin*
0.5 <0.03 <0.03 <0.03 <0.03
Genta- 6 2 <1 <1 <1
micin*
31582 -- G -- NG NG NG
Ampicillin*
0.5 -- <0.12 <0.12 <0.12
Penicillin
0.06 -- <0.03 <0.03 <0.03
Gentamicin
<1 -- <1 <1 <1
15972 -- G G NG NG NG
Ampicillin
<0.12 <0.12 <0.12 <0.12 <0.12
Penicillin
<0.03 <0.03 <0.03 <0.03 <0.03
Gentamicin
4 2 <1 <1 <1
Amikacin* 32 -- -- -- <16
015-035
-- G G NG NG NG
Ampicillin
<0.12 <0.12 <0.12 <0.12 <0.12
Penicillin
<0.03 <0.03 <0.03 <0.03 <0.03
Gentamicin
4 <1 <1 <1 <1
015-034
-- G -- NG NG NG
Ampicillin
<0.12 -- <0.12 <0.12 <0.12
Penicillin
<0.03 -- <0.03 <0.03 <0.03
Gentamicin
<1 -- <1 <1 <1
______________________________________
Minimum Inhibitory
Concentration of
Antibiotic (.mu.g/mL)
Organism With
Name With With 16
(Microspan .RTM.
Antibiotic 0 .mu.g/mL
4 .mu.g/mL
.mu.g/mL
ID No.) Tested rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
______________________________________
S. pnuemoniae
-- G NG NG
1293 Erythromycin* 1 <.25 <.25
Clindamycin** >2 <.25 <.25
Cefuroxime 4 <2 <2
Penicillin* 1 <.03 <.03
Chloramphenicol*
16 <4 <4
Gentamicin 4 <1 <1
Ampicillin 1 <.12 <.12
Norfloxacin** >8 <4 <4
Ciprofloxacin*
2 <1 <1
Amikacin* 32 <16 <16
S. pnuemoniae
-- G NG NG
130 Penicillin .06 <.03 <.03
Gentamicin* 6 <1 <1
Trimethoprim/ >2 <2 <2
Sulfamethoxazole**
Amikacin** >32 <16 <16
S. pnuemoniae
-- G NG NG
145 Cephalothin 4 <2 <2
Penicillin* 0.5 <.03 <.03
Gentamicin 4 <1 <1
Ampicillin 1 <.12 <.12
Trimethoprim/ >2 <2 <2
Sulfamethoxazole**
S. pnuemoniae
-- G NG NG
152 Gentamicin 4 <1 <1
Amikacin* 32 <16 <16
S. pnuemoniae
-- G G NG
154 Erythromycin* 1 <.25 <.25
Cefuroxime 4 <2 <2
Penicillin* 1 .12 <.03
Chlorampheicol*
16 8 <4
Gentamicin 4 <1 <1
Ampicillin 2 0.5 <.12
______________________________________
*BPI protein product reversed antibiotic indifference
**BPI protein product reversed antibiotic resistance
TABLE 4A
______________________________________
Susceptibility ranges for S. pnuemoniae
MIC (.mu.g/mL)
Antibiotic
Resistant Intermediate
Susceptible
______________________________________
Amikacin >32 32 .ltoreq.16
Ampicillin
.gtoreq.4 0.25-2 .ltoreq.0.12
Gentamicin
>6 6 .ltoreq.4
Penicillin
.gtoreq.2 0.12-1 .ltoreq.0.06
______________________________________
TABLE 4X
__________________________________________________________________________
EFFECTS OF BPI PROTEIN PRODUCTS .+-. ANTIBIOTICS
Minimum Inhibitory Concentration of Antibiotic (.mu.g/mL)
With:
Organism 4 16
Name 0 4 4 4 .mu.g/mL
16 16 16 .mu.g/mL
(Microspan .RTM.
Antibiotic
.mu.g/mL
.mu.g/mL
.mu.g/mL
.mu.g/mL
rBPI.sub.42
.mu.g/mL
.mu.g/mL
.mu.g/mL
rBPI.sub.42
ID No.)
Tested BPI rBPI.sub.23
rBPI.sub.21
BPI.sub.50
dimer
rBPI.sub.23
rBPI.sub.21
rBPI.sub.50
dimer
__________________________________________________________________________
S. -- G G NG G G G NG G NG
pneumoniae
Erythromycin*
1 <0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
1293 Clindamycin**
>2 <0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
<0.25
Cefuroxime
4 <2 <2 4 <2 <2 <2 <2 <2
Penicillin*
1 0.12
<0.03
0.5 <0.03
0.5 <0.03
0.5 <0.03
Chloramphenicol
16 <4 <4 8 <4 8 <4 <4 <4
Gentamicin
4 <1 <1 2 <1 4 <1 2 <1
Ampicillin
1 <0.12
<0.12
2 0.5 1 <0.12
1 1
Norfloxacin
>8 <4 <4 <4 <4 <4 <4 <4 <4
Trimethoprim/
>2 <2 <2 >2 <2 >2 <2 <2 <2
Sulfamethoxazole
Ciprofloxacin
2 <1 <1 2 <1 <1 <1 <1 <1
Amikacin 32 <16 <16 <16 <16 <16 <16 <16 <16
S -- G NG NG G NG G NG NG NG
pneumoniae
Penicillin
0.06
<0.03
<0.03
<0.03
<0.03
<0.03
<0.03
<0.03
<0.03
130 Gentamicin
6 <1 <1 6 <1 4 <1 <1 <1
Trimethoprim/
>2 <2 <2 <2 <2 >2 <2 <2 <2
Sulfamethoxazole
Amikacin >32 <16 <16 >32 <16 32 <16 <16 <16
S. -- G G NG G NG G NG G NG
pnuemoniae
Cefazolin
<2 <2 <2 <2 <2 4 <2 <2 <2
145 Cephalothin
4 <2 <2 <2 <2 4 <2 <2 <2
Penicillin*
0.5 0.06
<0.03
0.5 1 1 <0.03
<0.03
1
Gentamicin
4 <1 <1 2 <1 2 <1 <1 <1
Ampicillin
1 0.25
<0.12
0.5 0.5 2 <0.12
<0.12
2
Trimethoprim/
>2 <2 <2 <2 <2 >2 <2 <2 >2
Sulfamethoxazole
S. -- G G NG G G G NG G NG
pneumoniae
Gentamicin
4 4 <1 <1 2 4 <1 <1 <1
152 Amikacin*
32 <16 <16 32 <16 <16 <16 <16 <16
S. -- G G G G NG G NG G G
pneumoniae
Erythromycin*
1 <0.25
<0.25
0.5 <0.25
<0.25
<0.25
1 <0.25
154 Cefuroxime
4 <2 <2 4 <2 <2 <2 <2 <2
Penicillin*
1 0.25
0.12
1 <0.03
0.5 <0.03
0.5 0.25
Chloramphenicol*
16 8 8 8 <4 8 <4 8 <4
Gentamicin
4 2 <1 2 <1 4 <1 4 2
Ampicillin**
2 0.5 0.5 1 <0.12
2 <0.12
2 <0.12
Amikacin 32 <16 <16 32 <16 <16 <16 <16 <16
S. Penicillin
0.12
<0.03
<0.03
0.12
0.06
0.06
0.06
0.12
<.03
pneumoniae
Gentamicin
>6 >6 6 >6 >6 >6 >6 >6 6
141 Trimethoprim/
>2 <2 <2 >2 <2 <2 <2 <2 <2
Sulfa-
methoxazole**
Amikacin >32 >32 >32 >32 >32 >32 >32 >32 32
S. Cefazolin
4 <2 <2 4 <2 <2 <2 4 <2
pneumoniae
Cephalothin
4 <2 <2 4 <2 4 <2 4 <2
145 Penicillin*
0.5 <0.03
<0.03
0.5 <0.03
1 <0.03
0.5 <0.03
Gentamicin
4 4 <1 4 <1 6 <1 6 <1
Ampicillin**
1 <0.12
<0.12
2 <0.12
2 <0.12
2 <0.12
Trimethoprim/
>2 <2 <2 >2 <2 >2 <2 >2 <2
Sulfa-
methoxazole**
Amikacin*
32 <16 <16 <16 <16 32 <16 32 <16
__________________________________________________________________________
EXAMPLE 10
IN VITRO EFFECTS OF BPI PROTEIN PRODUCT ALOE OR IN COMBINATION WITH
ANTIBIOTICS ON STRAINS OF THE GRAM-POSITIVE ORGANISM STREPTOCOCCUS
PYOGENES (GROUP A)
The direct growth inhibitory effect of a BPI protein product, rBPI.sub.21,
on various strains of Streptococcus pyogenes, also known as Group A strep,
was evaluated using the Microscan.RTM. screening assay of Example 9. The
effect of rBPI.sub.21 on the antibiotic susceptibility of these strains
was also evaluated in the same assay. Assays were conducted on clinical
isolates of S. pyogenes (from Baxter Microscan.RTM. library, Sacramento,
Calif.).
A summary of the results of the BPI protein product and antibiotic
screening panels, reported as MICs (.mu.g/ml) of the tested antibiotic at
varying concentrations of rBPI.sub.21 .mu.g/ml), is shown in Table 5
below. Results are only reported where rBPI.sub.21 altered the antibiotic
susceptibility or growth. The antibiotic susceptibility standards
(interpretation of an MIC as clinically resistant, intermediate or
susceptible according to NCCLS standards) applicable to the organism
tested appear in Table 5A. Table 5 also shows the presence or absence of
bacterial growth ("G" or "NG") in the control well, which contains varying
concentrations of rBPI.sub.21 alone without antibiotic.
These results indicate that rBPI.sub.21, alone had a direct
bactericidal/growth inhibitory effect on one strain of S. pyogenes (Group
A) at a concentration as low as 2 .mu.g/ml, on three of the strains at 8
.mu.g/ml, and on the remaining strain at 32 .mu.g/ml. The data also show
that BPI protein product increased the antibiotic susceptibility of Strep.
pyogenes (Group A) strains to norfloxacin and gentamicin.
TABLE 5
__________________________________________________________________________
EFFECTS OF rBPI.sub.21 .+-. ANTIBIOTICS ON Streptococcus pyogenes (Group
A)
Minimum Inhibitory Concentration of Antibiotic (.mu.g/mL)
Microscan With With With With With With
Library
Antibiotic
0 .mu.g/mL
0.5 .mu.g/mL
2 .mu.g/mL
8 .mu.g/mL
16 .mu.g/mL
32 .mu.g/mL
ID No.
Tested rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
L rBPI.sub.21
__________________________________________________________________________
30847 -- G G G NG NG NG
Norfloxacin
<4 <4 <4 <4 <4 <4
Gentamicin
2 2 <1 <1 <1 <1
002-002
-- G G G G G NG
Norfloxacin
<4 <4 <4 <4 <4 <4
Gentamicin
2 <1 <1 <1 <1 <1
19035 -- G G G NG G NG
Norfloxacin
<4 <4 <4 <4 <4 <4
30403 -- G G NG NG NG NG
Norfloxacin*
8 <4 <4 <4 <4 <4
30413 -- G G G NG G NG
Norfloxacin*
8 <4 <4 <4 <4 <4
__________________________________________________________________________
*BPI protein product reversed antibiotic indifference
TABLE 5A
______________________________________
Susceptibility ranges for S. pyogenes (Group A)
MIC (.mu.g/mL)
Antibiotic
Resistant Intermediate
Susceptible
______________________________________
Gentamicin
>6 6 .ltoreq.4
Norfloxacin
>8 8 .ltoreq.4
______________________________________
EXAMPLE 11
IN VITRO EFFECTS OF BPI PROTEIN PRODUCT ALONE OR IN COMBINATION WITH
ANTIBIOTICS ON STRAINS OF THE GRAM-POSITIVE ORGANISM STREPTOCOCCUS
AGALACTIAE (GROUP B)
The effect of a BPI protein product, rBPI.sub.21, on the antibiotic
susceptibility of various strains of Streptococcus agalactiae, also known
as Group B strep, was evaluated using the Microscan.RTM. antibiotic
susceptibility screening assay of Example 9. The direct growth inhibitory
effect of rBPI.sub.21 on these strains was also evaluated in the same
assay. Assays were conducted on clinical isolates of S. agalactiae (from
Baxter Microscan.RTM. library, Sacramento, Calif.).
A summary of the results of the antibiotic screening panels, reported as
MICs (.mu.g/ml) of the antibiotic tested, is shown in Table 6 below.
Results are only reported where rBPI.sub.21 altered the antibiotic
susceptibility. The antibiotic susceptibility standards (interpretation of
an MIC as clinically resistant, intermediate or susceptible according to
NCCLS standards) applicable to the organism tested appear in Table 6A.
These results show that BPI protein product reversed the resistance of one
strain to gentamicin, and increased the susceptibility of other strains to
ciprofloxacin, gentamicin, norfloxacin. These strains were not susceptible
to BPI protein product alone, without antibiotic.
TABLE 6
__________________________________________________________________________
EFFECTS OF rBPI.sub.21 .+-. ANTIBIOTICS ON
Streptococcus agalactiae (Group B)
Minimum Inhibitory Concentration of Antibiotic (.mu.g/mL)
Microscan With With With With With
Library
Antibiotic
0 .mu.g/mL
0.5 .mu.g/mL
2 .mu.g/mL
8 .mu.g/mL
16 .mu.g/mL
ID No.
Tested rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
__________________________________________________________________________
003-044
Gentamicin*
>6 6 4 6 6
Ciprofloxacin
<1 <1 <1 <1 <1
Norfloxacin
<4 <4 <4 <4 <4
003-049
Gentamicin**
>6 4 4 4 4
Cipro- 2 2 <1 <1 <1
floxacin*
Norfloxacin
>8 8 8 8 8
30851 Gentamicin
4 <1 2 2 <1
Ciprofloxacin
<1 <1 <1 <1 <1
Norfloxacin*
8 <4 <4 <4 <4
31947 Gentamicin
2 4 <1 <1 <1
Cipro- 2 <1 <1 <1 <1
floxacin*
Norfloxacin
8 8 8 8 8
30911 Gentamicin
4 <1 4 <1 <1
Ciprofloxacin
<1 <1 <1 <1 <1
Norfloxacin
<4 <4 <4 <4 <4
__________________________________________________________________________
*BPI protein reversed antibiotic indifference
**BPI protein product reversed antibiotic resistance
TABLE 6A
______________________________________
Susceptibility ranges for S. agalactiae (Group B)
MIC (.mu.g/mL)
Antibiotic Resistant Intermediate
Susceptible
______________________________________
Ciprofloxacin
>2 2 .ltoreq.1
Gentamicin >6 6 .ltoreq.4
Norfloxacin
>8 8 .ltoreq.4
______________________________________
EXAMPLE 12
IN VITRO EFFECTS OF BPI PROTEIN PRODUCTS ALONE OR IN COMBINATION WITH
ANTIBIOTICS ON STRAINS OF THE GRAM-POSITIVE ORGANISM STREPTOCOCCUS BOVIS
The effect of a BPI protein product, rBPI.sub.21, on the antibiotic
susceptibility of various strains of Streptococcus bovis was evaluated
using the Microscan.RTM. antibiotic susceptibility screening assay of
Example 9. The direct growth inhibitory effect of rBPI21 on these strains
was also evaluated in the same assay. Assays were conducted on clinical
isolates of S. bovis (from Baxter Microscan.RTM. library, Sacramento,
Calif.).
A summary of the results of the antibiotic screening panels, reported as
MICs (.mu.g/ml) of the antibiotic tested, is shown in Table 7 below.
Results are only reported where rBPI.sub.21 altered the antibiotic
susceptibility. The antibiotic susceptibility standards (interpretation of
an MIC as clinically resistant, intermediate or susceptible according to
NCCLS standards) applicable to the organism tested appear in Table 7A.
These results show that the BPI protein product reversed the resistance of
one strain to ciprofloxacin, three strains to norfloxacin, and two strains
to tetracycline. The results show that BPI protein product also increased
the susceptibility of some strains to ciprofloxacin, gentamicin,
norfloxacin, and tetracycline. These strains were not susceptible to BPI
protein product alone, without antibiotic.
TABLE 7
__________________________________________________________________________
EFFECTS OF rBPI.sub.21 .+-. ANTIBIOTICS ON Streptococcus bovis
Minimum Inhibitory Concentration of Antibiotic (.mu.g/mL)
Microscan With With With With With
Library
Antibiotic
0 .mu.g/mL
0.5 .mu.g/mL
2 .mu.g/mL
8 .mu.g/mL
16 .mu.g/mL
ID No. Tested rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
__________________________________________________________________________
ATCC# Tetracycline*
128 8 8 4 8
49147 Gentamicin
2 2 <1 <1 <1
Cirpofloxacin
<1 <1 <1 <1 <1
Norfloxacin*
8 <4 <4 <4 <4
008-009
Tetracycline**
128 4 8 4 <2
Gentamicin
2 2 2 <1 <1
Ciprofloxacin*
2 <1 <1 <1 <1
Norfloxacin**
>8 8 8 8 <4
008-010
Tetracycline**
128 128 128 8 4
Gentamicin
4 4 2 2 <1
Ciprofloxacin*
2 2 <1 <1 <1
Norfloxacin**
>8 8 >8 <4 <4
008-011
Tetracycline
<2 <2 <2 <2 <2
Gentamicin
2 2 <1 <1 <1
Ciprofloxacin*
2 <1 2 <1 <1
Norfloxacin**
>8 8 8 8 <4
008-012
Tetracycline
128 128 128 128 8
Gentamicin
4 2 <1 2 <1
Ciprofloxacin**
>2 2 <1 2 <1
Norfloxacin
>8 8 8 8 8
__________________________________________________________________________
*BPI protein product reversed antibiotic indifference
**BPI protein product reversed antibiotic resistance
TABLE 7A
______________________________________
Susceptibility ranges for S. bovis
MIC (.mu.g/mL)
Antibiotic Resistant Intermediate
Susceptibe
______________________________________
Ciprofloxacin
>2 2 .ltoreq.1
Gentamicin >6 6 .ltoreq.4
Norfloxacin
>8 8 .ltoreq.4
Tetracycline
.gtoreq.128
8 .ltoreq.4
______________________________________
EXAMPLE 13
IN VITRO EFFECTS OF BPI PROTEIN PRODUCTS ALONE OR IN COMBINATION WITH
ANTIBIOTICS ON STRAINS OF THE GRAM-POSITIVE ORGANISM ENTEROCOCCUS FAECALIS
The effect of a BPI protein product, rBPI.sub.21, on the antibiotic
susceptibility of various strains of Enterococcus faecalis was evaluated
using the Microscan.RTM. antibiotic susceptibility screening assay of
Example 9. The direct growth inhibitory effect of rBPI.sub.21 on these
strains was also evaluated in the same assay. Assays were conducted on
clinical isolates of E. faecalis (from Baxter Microscan.RTM. library,
Sacramento, Calif.).
A summary of the results of the antibiotic screening panels, reported as
MICs (.mu.g/ml) of the antibiotic tested, is shown in Table 8 below.
Results are only reported where rBPI.sub.21 altered the antibiotic
susceptibility. The antibiotic susceptibility standards (interpretation of
an MIC as clinically resistant, intermediate or susceptible according to
NCCLS standards) applicable to the organism tested appear in Table 8A.
These results show that BPI protein product reversed the resistance of
three strains to ciprofloxacin and two strains to norfloxacin. The BPI
protein product also increased the susceptibility of some E. faecalis
strains to ampicillin, ciprofloxacin, erythromycin, noffloxacin,
penicillin, rifampin, and tetracycline. These strains were not susceptible
to BPI protein product alone, without antibiotic.
TABLE 8
__________________________________________________________________________
EFFECTS OF rBPI.sub.21 .+-. ANTIBIOTICS ON Enterococcus faecalis
Minimum Inhibitory Concentration of Antibiotic
(.mu.g/mL)
Microscan With With With With
Library
Antibiotic
0 .mu.g/mL
2 .mu.g/mL
8 .mu.g/mL
32 .mu.g/mL
ID No. Tested rBPI.sup.21
rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
__________________________________________________________________________
011-070
Penicillin
2 2 1 1
Tetracycline
128 128 128 8
16967 Rifampin
>2 >2 >2 2
Ciprofloxacin**
>2 2 2 <1
Norfloxacin**
>8 8 8 <4
Penicillin
2 2 2 1
Erythromycin
4 2 2 2
19010 Pencillin
8 8 8 4
Ampicillin
4 4 4 2
011-028
Penicillin
2 2 2 1
Ciprofloxacin*
2 <1 <1 <1
Ampicillin
1 <0.5 <0.5 <0.5
011-001
Ciprofloxacin**
>2 <1 <1 <1
Norfloxacin
<4 <4 <4 <4
011-002
Penicillin
2 1 1 1
Ampicillin
2 0.5 0.5 0.5
011-003
Rifampin
>2 >2 >2 2
Penicillin
2 2 2 1
Norfloxacin*
8 8 8 <4
Ampicillin
1 1 0.5 0.5
011-004
Penicillin
8 8 8 4
Ampicillin
4 4 2 2
011-005
Penicillin
2 2 2 1
Ampicillin
1 0.5 0.5 0.5
011-006
Penicillin
2 2 2 1
Ampicillin
1 1 1 0.5
011-066
Ciprofloxacin**
>2 <1 <1 <1
Norfloxacin**
>8 <4 <4 <4
Ampicillin
1 1 0.5 0.5
__________________________________________________________________________
*BPI protein product reversed antibiotic indifference
**BPI protein product reversed antibiotic resistance
TABLE 8A
______________________________________
Susceptibility ranges for E. faecalis
MIC (.mu.g/mL)
Antibiotic Resistant Intermediate
Susceptible
______________________________________
Ampicillin >8 .ltoreq.8
Ciprofloxacin
>2 2 .ltoreq.1
Erythromycin
>4 1-4 .ltoreq.0.5
Norfloxacin
>8 8 .ltoreq.4
Penicillin >8 .ltoreq.8
Rifampin >2 2 .ltoreq.1
Tetracycline
.gtoreq.128
8 .ltoreq.4
______________________________________
EXAMPLE 14
IN VITRO EFFECTS OF BPI PROTEIN PRODUCTS ALONE OR IN COMBINATION WITH
ANTIBIOTICS ON STRAINS OF THE GRAM-POSITIVE ORGANISM ENTEROCOCCUS FAECIUM
The effect of a variety of BPI protein products on the antibiotic
susceptibility of various strains of Enterococcus faecium was evaluated
using the Microscan.RTM. antibiotic susceptibility screening assay of
Example 9. The direct growth inhibitory effect of the BPI protein products
on these strains was also evaluated in the same assay. Assays were
conducted on clinical isolates of E. faecium (from Baxter Microscan.RTM.
library, Sacramento, Calif.).
A summary of the results of the antibiotic screening panels, reported as
MICs .mu.g/ml) of the antibiotic tested at varying concentrations of
rBPI.sub.21, is shown in Table 9 below. Results are only reported where
rBPI.sub.21, altered the antibiotic susceptibility. The antibiotic
susceptibility standards (interpretation of an MIC as clinically
resistant, intermediate or susceptible according to NCCLS standards)
applicable to the organism tested appear in Table 9A. These results show
that rBPI.sub.21, reversed the resistance of five strains to amoxicillin/K
clavulanate, two strains to cefazolin, two strains to ciprofloxacin, two
strains to erythromycin, one strain to norfloxacin, two strains to
oxacillin, two strains to rifampin, one strain to tetracycline, and one
strain to vancomycin. The rBPI.sub.21 also increased the susceptibility of
some E. faecium strains to amoxicillin/K clavulanate, cefotaxime,
ciprofloxacin, erythromycin, penicillin, rifampin, and vancomycin. These
strains were not susceptible to BPI protein product alone, without
antibiotic.
Additional screening of an E. faecium strain (Microscan.RTM. ID no. 15773)
was conducted with a variety of BPI protein products, rBPI.sub.21,
rBPI.sub.23, rBPI.sub.50 and rBPI.sub.42 dimer. Table 9X below displays a
summary of the results of the antibiotic screening panels, reported as the
MIC of the tested antibiotics at varying concentrations of the indicated
BPI protein product. Results are only reported if antibiotic
susceptibility was altered.
TABLE 9
__________________________________________________________________________
EFFECTS ON rBPI.sub.21 .+-. ANTIBIOTICS ON Enterococcus faecium
Minimum Inhibitory Concentration of Antibiotic
(.mu.g/mL)
Microscan With With With With
Library
Antibiotic
0 .mu.g/mL
2 .mu.g/mL
8 .mu.g/mL
32 .mu.g/mL
ID No. Tested rBPI.sup.21
rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
__________________________________________________________________________
17611 Rifampin**
>2 >2 2 <1
Ciprofloxacin**
>2 2 2 <1
Augmentin.sup.a
>16 16 16 16
16866 Ciprofloxacin*
2 2 <1 <1
Tetracycline**
128 128 128 4
20076 Rifampin**
>2 2 2 <1
Erythromycin*
4 2 1 <0.25
Augmentin.sup.a **
16 8 8 <4
Oxacillin**
>4 <0.5 <0.5 <0.5
012-048
Rifampin
>2 >2 2 2
Ciprofloxacin**
>2 >2 <1 <1
Penicillin
0.25 0.25 0.25 0.12
Cefotaxime*
32 <8 <8 <8
Cefazolin**
>16 16 8 8
15773 Rifampin*
2 2 <1 <1
Ciprofloxacin*
2 <1 <1 <1
Erythromycin**
>4 <0.25 <0.25 <0.25
Augmentin.sup.a **
16 <4 <4 <4
Vancomycin
4 4 <2 <2
012-001
Rifampin
>2 2 >2 2
Augmentin.sup.a **
16 8 8 8
012-002
Rifampin
>2 >2 >2 2
Oxacillin**
>4 1 <0.5 <0.5
Vancomycin**
>16 >16 >16 <2
Norfloxacin**
>8 <4 <4 <4
Cefazolin**
>16 >16 >16 8
012-003
Rifampin
<1 <1 <1 <1
Augmentin.sup.a **
16 8 8 8
012-004
Rifampin*
2 2 <1 <1
Ciprofloxacin*
2 <1 <1 <1
Augmentin.sup.a **
16 <4 <4 <4
Vancomycin
4 4 <2 <2
Erythromycin**
>4 0.5 <0.25 <0.25
__________________________________________________________________________
.sup.a Augmentin is Amoxicillin K/Clavulanate
*BPI protein product reversed antibiotic indifference
**BPI protein product reversed antibiotic resistance
TABLE 9A
______________________________________
Susceptibility ranges for E. faecium
MIC (.mu.g/mL)
Antibiotic Resistant Intermediate
Susceptible
______________________________________
Amoxicillin .gtoreq.16/8 .ltoreq.8/4
K/Clavulanate
Cefazolin >16 16 .ltoreq.8
Cefotaxime >32 16-32 .ltoreq.8
Ciprofloxacin
>2 2 .ltoreq.1
Erythromycin
>4 1-4 .ltoreq.0.5
Norfloxacin >8 8 .ltoreq.4
Oxacillin .gtoreq.4 .ltoreq.2
(staph only)
Penicillin >8 .ltoreq.8
Rifampin >2 2 .ltoreq.1
Tetracycline
.gtoreq.128
8 .ltoreq.4
Vancomycin >16 8-16 .ltoreq.4
______________________________________
TABLE 9X
__________________________________________________________________________
EFFECTS OF BPI PROTEIN PRODUCTS .+-. ANTIBIOTICS
Minimum Inhibitory Concentration of Antibiotic
(.mu.g/mL) With:
Organism 4 16
Name 0 4 4 4 .mu.g/mL
16 16 16 .mu.g/mL
(Microscan .RTM.
Antibiotic
.mu.g/mL
.mu.g/mL
.mu.g/mL
.mu.g/mL
rBPI.sub.42
.mu.g/mL
.mu.g/mL
.mu.g/mL
rBPI.sub.42
ID No.)
Tested BPI rBPI.sub.21
rBPI.sub.23
rBPI.sub.50
dimer
rBPI.sub.21
rBPI.sub.23
rBPI.sub.50
dimer
__________________________________________________________________________
E. faecium
Erythromycin**
>4 1 <0.25
>4 >4 <0.25
<0.25
>4 <0.25
15773 Vancomycin
4 4 4 4 4 <2 4 4 <2
Cefotaxime
>32 >32 >32 >32 >32 >32 32 >32 >32
Ticarcillin/K
>8 >8 >8 >8 >8 >8 8 >8 >8
Clavulanate
Gentamicin**
>6 4 4 >6 >6 6 6 >6 4
Amoxicillin/K
16 8 <4 16 8 <4 <4 8 <4
Clavulanate
Ampicillin/
16 <8 <8 16 <8 <8 <8 <8 <8
Sulbactam
Rifampin >2 2 2 >2 2 <1 <1 >2 <1
Ciprofloxacin*
2 <1 <1 2 <1 <1 <1 <1 <1
Amikacin* 32 <16 <16 32 32 <16 32 32 <16
Sulfamethoxazole**
>256
<256
<256 >256
>256
<256 >256 >256
<256
__________________________________________________________________________
EXAMPLE 15
IN VITRO EFFECTS OF BPI PROTEIN PRODUCTS ALONE OR IN COMBINATION WITH
ANTIBIOTICS ON OTHER GRAM-POSITIVE ENTEROCOCCUS SPECIES
The effect of a BPI protein product, rBPI.sub.21, on the antibiotic
susceptibility of a variety of Enterococcus species was evaluated using
the Microscan.RTM. antibiotic susceptibility screening assay of Example 9.
The direct growth inhibitory effect of rBPI.sub.21 on these species was
also evaluated in the same assay. Assays were conducted on clinical
isolates of Enterococcus species (from Baxter Microscan.RTM. library,
Sacramento, Calif.).
A summary of the results of the antibiotic screening panels, reported as
MICs (.mu.g/ml) of the antibiotic tested, is shown in Table 10 below.
Results are only reported where rBPI.sub.21 altered the antibiotic
susceptibility. The antibiotic susceptibility standards (interpretation of
an MIC as clinically resistant, intermediate or susceptible according to
NCCLS standards) applicable to the organism tested appear in Table 10A.
These results show that for E. gallinarum, BPI protein product reversed
resistance to cephalothin, cefazolin, ciprofloxacin and norfloxacin, and
increased susceptibility to ampicillin, penicillin and rifampin. For E.
raffinosus, BPI protein product reversed resistance to ampicillin and
increased susceptibility to imipenem. For E. casseliflavus, BPI protein
product increased susceptibility to ampicillin, penicillin, oxacillin,
cephalothin, and cefazolin. For E. durans, BPI protein product increased
susceptibility to ampicillin and rifampin. For E. avium, BPI protein
product increased susceptibility to ampicillin, penicillin, cefotaxime and
ciprofloxacin, and oxacillin. These Enterococcus species were not
susceptible to BPI protein product alone, without antibiotic.
TABLE 10
______________________________________
EFFECTS OF rBPI.sub.21 .+-.
ANTIBIOTICS ON Enterococus SPECIES
Minimum Inhibitory Concen-
tration of Antibiotic (.mu.g/mL)
With With With With
Microscan 0 2 8 32
Library Antibiotic .mu.g/mL
.mu.g/mL
.mu.g/mL
.mu.g/mL
ID No. Tested rBPI.sup.21
rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
______________________________________
031-009 Ampicillin 1 1 0.5 0.5
( E. Penicillin 1 0.5 0.5 0.25
gallinarum)
Rifampin >2 >2 >2 2
Cephalothin**
>16 <8 <8 <8
Cefazolin** >16 8 8 4
Ciprofloxacin**
>2 2 <1 <1
Norfloxacin**
>8 8 8 <4
16206 Ampicillin**
>8 >8 >8 8
(E. raffinosus)
Imipenem >8 >8 >8 8
030-008 and
Ampicillin 1 0.5 0.25 0.25
030-001.sup.a
Penicillin 1 0.25 0.25 0.12
( E. Oxacillin >4 >4 >4 4
casseliflavus)
Cephalothin*
16 <8 <8 <8
Cefazolin* 16 8 4 4
17543 Ampicillin 1 0.5 0.5 0.5
(E. durans)
Rifampin >2 2 2 2
31413 Ampicillin 0.5 0.5 0.5 <0.12
(E. avium)
Penicillin 1 1 1 0.5
Oxacillin** >4 >4 4 0.5
Cefotaxime* 32 <8 <8 <88
Ciprofloxacin*
2 <1 <1 <1
______________________________________
.sup.1 Two strains of E. casseliflavus were tested; both strains gave
identical results.
TABLE 10A
______________________________________
Susceptibility ranges for Enterococcus species
MIC (.mu.g/mL)
Antibiotic Resistant Intermediate
Susceptible
______________________________________
Ampicillin >8 .ltoreq.8
Cefazolin >16 16 .ltoreq.8
Cefotaxime >32 16-32 .ltoreq.8
Cephalothin
>16 16 .ltoreq.8
Ciprofloxacin
>2 2 .ltoreq.1
Imipenem >8 8 .ltoreq.4
Norfloxacin
>8 8 .ltoreq.4
Oxacillin .gtoreq.4 .ltoreq.2
Penicillin >8 .ltoreq.8
Rifampin >2 2 .ltoreq.1
______________________________________
EXAMPLE 16
IN VITRO EFFECTS OF BPI PROTEIN PRODUCTS ALONE OR IN COMBINATION WITH
ANTIBIOTICS ON STRAINS OF THE GRAM-POSITIVE ORGANISM STAPHYLOCOCCUS AUREUS
The effect of BPI protein products on the antibiotic susceptibility of a
variety of S. aureus species was evaluated using the Microscan.RTM.
antibiotic susceptibility screening assay of Example 9. The direct growth
inhibitory effect of BPI protein products on these species was also
evaluated in the same assay. Assays were conducted on the following
clinical isolates of S. aureus (from Baxter Microscan.RTM. library,
Sacramento, Calif.): 052-066, 052-106, 052-107, 052-108, 052-184, 052-219,
052-230, 14288, 20720, 29213 (ATCC No.), 32075 and 32073.
These strains of S. aureus were tested to determine the MIC.sub.90 for each
antibiotic in the panel at varying concentrations of rBPI.sub.21. The
MIC.sub.90 is defined as the lowest concentration of antibiotic that
inhibits the growth of 90% of all S. aureus isolates tested. Results are
reported in Table 11 below, and applicable antibiotic susceptibility
standards (interpretation of an MIC as clinically resistant, intermediate
or susceptible according to NCCLS standards) appear in Table 11A.
The data show that rBPI.sub.21 was able to reduce the S. aureus MIC.sub.90
values for eight of twenty-five antibiotics evaluated: amoxicillin/K
clavulanate (Augmentin), cefotaxime, ceftriaxone, cefuroxime, cephalothin,
chloramphenicol, imipenem and sulfamethoxazole. Dashes in Table 11
indicate that no concentration of antibiotic tested was able to inhibit
growth of 90% of all tested strains. None of the S. aureus strains tested
were susceptible to rBPI.sub.21 alone without antibiotic.
Comparable results obtained from testing of the BPI protein product
rBPI.sub.21 on S. aureus (Microscan.RTM. ID no. 052-106) in the Pasco
system confirm the effect of BPI protein products on the antibiotic
susceptibility of gram-positive organisms.
Additional screening of this S. aureus strain (Microscan.RTM.ID no.
052-106) was conducted with a variety of BPI protein products,
rBPI.sub.21, rBPI.sub.23, rBPI.sub.50 and rBPI.sub.42 dimer. Table 11X
below displays a summary of the results of the antibiotic screening
panels, reported as the MIC of the tested antibiotics at varying
concentrations of the indicated BPI protein product. Results are only
reported if antibiotic susceptibility was altered.
TABLE 11
______________________________________
Effect of rBPI.sub.21 on MIC.sub.90 of Antibiotics for Staphylococcus
aureus
MIC.sub.90 With
MIC.sub.90 With
MIC.sub.90 With
0 .mu.g/mL 0.5-8 .mu.g/mL
16-64 .mu.g/mL
rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
Antibiotic Tested
(n = 38).sup.a
(n = 31).sup.a
(n = 25).sup.a
______________________________________
Amikacin 16 16 16
Amox/K Clav 16 16 8
Amp/Sulbactam
8 8 8
Ampicillin -- -- --
Cefazolin -- -- --
Cefataxime 32 8 4
Ceftriaxone -- 32 8
Cefuroxime -- -- 16
Cephalothin 8 4 4
Chloramphenicol
8 8 4
Ciprofloxacin
1 1 1
Clindamycin -- -- --
Erythromycin
-- -- --
Gentamicin -- -- --
Imipenem -- -- 8
Nitrofurantoin
32 32 32
Norfloxacin 4 4 4
Oxacillin -- -- --
Penicillin -- -- --
Rifampin 1 1 1
Sulfamethoxazole
-- -- 256
Tetracycline
128 128 128
Ticarcillin/K Clav
-- -- --
Trimeth/Sulfa
2 2 2
Vancomycin 2 2 2
______________________________________
.sup.a Number of runs; 12 strains tested
TABLE 11A
______________________________________
Susceptibility ranges for S. aureus
MIC (.mu.g/mL)
Antibiotic Resistant Intermediate
Susceptible
______________________________________
Amikacin >32 32 .ltoreq.16
Amoxicillin/K Clav.
.gtoreq.8/4 .ltoreq.4/2
Ampicillin/Sulbactam
>16/8 16/8 .ltoreq.8/4
Ampicillin .gtoreq.0.5 .ltoreq.0.25
Cefazolin >16 16 .ltoreq.8
Cefotaxime >32 16-32 .ltoreq.8
Ceftriaxone >32 16-32 .ltoreq.8
Cefuroxime >16 16 .ltoreq.8
Cephalothin >16 16 .ltoreq.8
Chloramphenicol
>16 16 .ltoreq.8
Ciprofloxacin
>2 2 .ltoreq.1
Clindamycin >2 1-2 .ltoreq.0.5
Erythromycin >4 1-4 .ltoreq.0.5
Gentamicin >6 6 .ltoreq.4
Imipenem >8 8 .ltoreq.4
Nitrofurantoin
>64 64 .ltoreq.32
Norfloxacin >8 8 .ltoreq.4
Oxacillin .gtoreq.4 .ltoreq.2
Penicillin .gtoreq.0.25 .ltoreq.0.12
Rifampin >2 2 .ltoreq.1
Sulfamethoxazole
>256 .ltoreq.256
Tetracycline .gtoreq.128
8 .ltoreq.4
Ticarcillin/K Clav.
.gtoreq.8 .ltoreq.4
Trimethoprim/
>2/38 .ltoreq.2/38
Surfamethoxazole
Vancomycin >16 8-16 .ltoreq.4
______________________________________
TABLE 11X
__________________________________________________________________________
EFFECTS OF BPI PROTEIN PRODUCTS .+-. ANTIOBIOTICS
Minimum Inhibitory Concentration of Antibiotic
(.mu.g/mL) With:
Organism 4 16
Name 0 4 4 4 .mu.g/mL
16 16 16 .mu.g/mL
(Microscan .RTM.
Antibiotic
.mu.g/mL
.mu.g/mL
.mu.g/mL
.mu.g/mL
rBPI.sub.42
.mu.g/mL
.mu.g/mL
.mu.g/mL
rBPI.sub.42
ID No.)
Tested BPI rBPI.sub.21
rBPI.sub.23
rBPI.sub.50
dimer
rBPI.sub.21
rBPI.sub.23
rBPI.sub.50
dimer
__________________________________________________________________________
S. aureus
Cephalothin
8 4 4 4 8 4 4 4 4
052-106
Cefuroxime**
>16 16 16 16 >16 8 8 8 8
Ceftriaxone*
16 8 8 8 16 8 8 8 8
Chloramphenicol
8 8 8 8 8 <4 <4 16 <4
Trimethoprim/
>2 <2 <2 <2 <2 <2 <2 <2 <2
Sulfamethoxazole**
Ciprofloxacin
<1 <1 <1 <1 <1 <1 <1 >2 <1
Sulfamethoxazole**
>256
>256
>256
>256
>256
<256
>256
>256
<256
__________________________________________________________________________
EXAMPLE 17
IN VITRO EFFECTS OF BPI PROTEIN PRODUCTS ALONE OR IN COMBINATION WITH
ANTIBIOTICS ON STRAINS OF THE GRAM-POSITIVE ORGANISM STAPHYLOCOCCUS
EPIDERMIDIS
The effect of a BPI protein product, rBPI.sub.21, on the antibiotic
susceptibility of a variety of Staphylococcus epidermidis species was
evaluated using the Microscan.RTM. antibiotic susceptibility screening
assay of Example 9. The direct growth inhibitory effect of rBPI.sub.21 on
these species was also evaluated in the same assay. Assays were conducted
on the following clinical isolates of S. epidermidis (from Baxter
Microscan.RTM. library, Sacramento, Calif.): 055051, 055155, 19776, 20778,
20959, 055125, 055129, 055126, 32086 and 32085. S. epidermidis is a
coagulase-negative staphylococcus that is a major agent of nosocomial
(hospital-acquired) sepsis in oncology and neonatal sections, and accounts
for about 40% of all prosthetic joint infections.
These strains of S. epidemidis were tested to determine the MIC.sub.90 for
each antibiotic in the panel at varying concentrations of rBPI.sub.21.
Results are reported in Table 12 below, and applicable antibiotic
susceptibility standards (interpretation of an MIC as clinically
resistant, intermediate or susceptible according to NCCLS standards)
appear in Table 12A.
The data show that the BPI protein product was able to reduce the S.
epidermidis MIC.sub.90 values for thirteen of twenty-five antibiotics
evaluated: amikacin, amoxicillin/K clavulanate (augmentin), ampicillin,
cefazolin, cefotaxime, ceftriaxone, cefuroxime, chloramphenicol,
penicillin, sulfamethoxazole, tetracycline, ticarcillin/K clavulanate and
trimethoprim/sulfamethoxazole. None of the S. epidermidis strains tested
were susceptible to BPI protein product alone, without antibiotic.
TABLE 12
______________________________________
Effect of rBPI.sub.21 on MIC.sub.90 of Antibiotics
for Staphylococcus epidermidis
MIC.sub.90 With
MIC.sub.90 With
MIC.sub.90 With
0 .mu.g/mL 8 .mu.g/mL 32 .mu.g/mL
rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
Antibiotic Tested
(n = 35).sup.a
(n = 10).sup.a
(n = 9).sup.a
______________________________________
Amikacin 32 NT 16
Amoxicillin/K Clav
8 8 4
Ampicillin/ 8 NT 8
Sulbactam
Ampicillin -- -- 4
Cefazolin 4 4 2
Cefotaxime -- -- 8
Ceftriaxone -- NT 4
Cefuroxime -- NT 2
Cephalothin 8 8 8
Chloramhenicol
-- NT 4
Ciprofloxacin
1 1 1
Clindamycin -- -- --
Erythromycin
-- -- --
Gentamicin -- -- --
Imipenem -- -- --
Nitrofurantoin
32 32 32
Norfloxacin 4 4 4
Oxacillin -- -- --
Penicillin -- -- 8
Rifampin 1 1 1
Sulfamethoxazole
-- NT 256
Tetracycline
128 128 8
Ticarcillin/K Clav
-- NT 1
Trimeth/Sulfa
-- 2 2
Vancomycin 2 2 2
______________________________________
.sup.a Number of runs.
TABLE 12A
______________________________________
Susceptibility ranges for S. epidermidis
MIC (.mu.g/mL)
Antibiotic Resistant Intermediate
Susceptible
______________________________________
Amikacin >32 32 .ltoreq.16
Amoxicillin/K Clav.
.gtoreq.8/4 .ltoreq.4/2
Ampicillin/Sulbactam
>16/8 16/8 .ltoreq.8/4
Ampicillin .gtoreq.0.5 .ltoreq.0.25
Cefazolin >16 16 .ltoreq.8
Cefotaxime >32 16-32 .ltoreq.8
Ceftriaxone >32 16-32 .ltoreq.8
Cefuroxime >16 16 .ltoreq.8
Cephalothin >16 16 .ltoreq.8
Chloramphenicol
>16 16 .ltoreq.8
Ciprofloxacin
>2 2 .ltoreq.1
Clindamycin >2 1-2 .ltoreq.0.5
Erythromycin >4 1-4 .ltoreq.0.5
Gentamicin >6 6 .ltoreq.4
Imipenem >8 8 .ltoreq.4
Nitrofurantoin
>64 64 .ltoreq.32
Norfloxacin >8 8 .ltoreq.4
Oxacillin .gtoreq.4 .ltoreq.2
Penicillin .gtoreq.0.25 .ltoreq.0.12
Rifampin >2 2 .ltoreq.1
Sulfamethoxazole
>256 .ltoreq.256
Tetracycline .gtoreq.128
8 .ltoreq.4
Ticarcillin/K Clav.
.gtoreq.8 .ltoreq.4
Trimethoprim/
>2/38 .ltoreq.2/38
Surfamethoxazole
Vancomycin >16 8-16 .ltoreq.4
______________________________________
EXAMPLE 18
IN VITRO EFFECTS OF BPI PROTEIN PRODUCTS ALONE OR IN COMBINATION WITH
ANTIBIOTICS ON SPECIES OF THE GRAM-POSITIVE ORGANISM STAPHYLOCOCCUS
The effect of a BPI protein product, rBPI.sub.21, on the antibiotic
susceptibility of a variety of other coagulase-negative Staphylococcus
species was evaluated using the Microscan.RTM. antibiotic susceptibility
screening assay of Example 9. The direct growth inhibitory effect of
rBPI.sub.21 on these species was also evaluated in the same assay. Assays
were conducted on clinical isolates of Staphylococcus species (from Baxter
Microscan.RTM. library, Sacramento, Calif.). In the last decade there has
been a marked increase in clinical infections caused by the
coagulase-negative staphylococci. These organisms are also significant
opportunistic pathogens.
A summary of the results of the antibiotic screening panels, reported as
MICs (.mu.g/ml) of the antibiotic tested, is shown in Table 13 below. The
antibiotic susceptibility standards (interpretation of an MIC as
clinically resistant, intermediate or susceptible according to NCCLS
standards) applicable to the organism tested appear in Table 13A.
These results show that BPI protein product reversed resistance to
penicillin for one S. hominis strain, and reversed resistance to
trimethoprim/sulfamethoxazole for one S. haemolyticus strain and the S.
intermedius strain. BPI protein product increased susceptibility of S.
hominis strains to ampicillin and penicillin, the S. sciuri strain to
clindamycin and penicillin, the S. saprophyticus strain to erythromycin,
S. haemolyticus strains to erythromycin, and the S. hyicus strain to
clindamycin and erythromycin, the S. intermedius strain to erythromycin,
and the S. simulans strain to erythromycin. None of these tested strains
were susceptible to BPI.sub.21 alone at the concentrations tested.
TABLE 13
______________________________________
Effects of rBPI.sub.21 on Staphylococcus Species
Minimum Inhibitory
Concentration (.mu.g/ml)
With 0 With 4
With 16
Microscan Antibiotic .mu.g/ml .mu.g/ml
.mu.g/ml
Library ID No..sup.a
Tested.sup.b
rBPI.sub.21
rBPI.sub.21
rBPI.sub.21
______________________________________
( S. hominis)
Ampicillin 0.25 <0.12 <0.12
057-003 Penicillin**
0.5 0.06 0.06
(S. hominis)
Ampicillin 0.25 0.25 <0.12
057-001 Penicillin 0.25 0.12 0.06
(S. hominis)
Ampicillin 0.25 0.5 0.25
057-024 Penicillin 0.5 0.5 0.5
(S. scirui) Pencillin 0.06 0.06 <0.03
060-016 Clindamycin*
1 1 0.5
(S. saprophyticus)
Erythromycin
0.5 0.5 <0.25
059-003
(S. haemolyticus)
Trim/Sulfa**
>2 <2 <2
19770 Erythromycin
>4 4 4
(S. haemolyticus)
Ampicillin 0.5 1 0.5
056-042 Penicillin 0.25 0.5 0.25
(S. hyicus) Erythromycin
0.5 <0.25 <0.25
10377 Clindamycin
0.5 <0.25 <0.25
(S. intermedius)
Trim/Sulfa**
>2 <2 <2
10254 Erythromycin
0.5 <0.25 <0.25
(S. simulans)
Erythromycin
0.5 0.5 <0.25
061-001
______________________________________
.sup.a Two strains of S. lugdunensis (Microscan library ID Nos. 19782 and
11130) were also tested but were susceptible to all antibiotics tested.
.sup.b Strains were susceptible to all other antibiotics that were tested
but are not shown here.
TABLE 13A
______________________________________
Susceptibility ranges for Staphylococcus species
MIC (.mu.g/mL)
Antibiotic Resistant Intermediate
Susceptible
______________________________________
Ampicillin .gtoreq.0.5 .ltoreq.0.25
Clindamycin >2 1-2 .ltoreq.0.5
Erythromycin >4 1-4 .ltoreq.0.5
Penicillin .gtoreq.0.25 .ltoreq.0.12
Trimethoprim/
>2/38 .ltoreq.2/38
Sulfamethoxazole
______________________________________
EXAMPLE 19
EARLY IN VITRO BACTERICIDAL EFFECTS OF BPI PROTEIN PRODUCT ALONE OR IN
COMBINATION WITH ANTIBIOTICS ON S. AUREUS, S. PNEUMONIAE, AND E. FAECIUM
The effect of a BPI protein product, rBPI.sub.21, on the killing curves of
selected antibiotics was determined for selected organisms. Microscan.RTM.
panel plates were prepared for a methicillin-resistant S. aureus
(Microscan library ID no. 052-106), S. pneumoniae (Microscan library ID
no. 31573), and E. faecium (Microscan library ID no. 15773), according to
Example 9. Cell suspensions were added to 25 ml Pluronic Inoculum Water
containing 0 or 16 .mu.g/ml rPBI.sub.21. After inoculation, the panel
plates were incubated at 35.degree. C. for 24 hours. At 0, 4, 7 and 24
hours after inoculation, 5 .mu.l samples were removed from each growth
control well (containing culture media without antibiotic) and from each
well containing: 8 .mu.g/ml penicillin, 2 .mu.g/ml ciprofloxacin, 256
.mu.g/ml sulfamethoxazole, 32 .mu.g/ml cefotaxime, 16 .mu.g/ml
chloramphenicol, or 16 .mu.g/ml vancomycin. These 5 .mu.l samples were
diluted in sterile water and inoculated onto Trypticase Soy agar plates
(Remel, Lenexa, Kans.) or blood agar plates (Remel, Lenexa, Kans.) for S.
pneumoniae. After 48 hours of incubation at 35.degree. C., the plates were
counted and the number of colony forming units of bacteria remaining in
the well was calculated.
The results are shown below in FIGS. 26 through 32. In all of the figures,
the growth in the presence of antibiotic alone (without rPBI.sub.21) is
indicated for: S. aureus (a filled square), S. pneumoniae (a fried
diamond) or E. faecium (a filled triangle). Also, in all figures, the
growth in the presence of antibiotic with rPBI.sub.21 is indicated for: S.
aureus (an open square), S. pneumoniae (an open diamond) or E. faecium (an
open triangle).
FIG. 26 shows the growth curve of organisms with rBPI.sub.21 (and without
antibiotic) and without rPBI.sub.21 (and without antibiotic). In FIG. 26,
the growth curves for S. aureus with rBPI.sub.21 (open squares) and E.
faecium without rBPI.sub.21 (filled triangles) overlap at 7 and 24 hours.
The results show that BPI protein product has a dramatic bactericidal
effect on S. pneumoniae that continues through 24 hours, and a moderate
early inhibitory effect on growth of E. faecium that is not sustained
after 10 hours.
In FIG. 27, the growth curves for S. pneumoniae with rPBI.sub.21 (open
diamonds) and S. pneumoniae without rBPI.sub.21 (filled diamonds) overlap
at 7 and 24 hours. FIG. 27 shows that the BPI protein product enhanced the
early bactericidal effect of penicillin on all three organisms at 0-10
hours.
In FIG. 28, the growth curves for S. pneumoniae with rPBI.sub.21 (open
diamonds) and S. pneumoniae without rPBI.sub.21 (filled diamonds) overlap
completely. FIG. 28 shows that BPI protein product enhanced the early
bactericidal activity of cefotaxime for S. aureus and E. faecium at 0-10
hours.
FIG. 29 shows that BPI protein product enhanced the early bactericidal
activity of chloramphenicol for all three organisms at 0-10 hours.
FIG. 30 shows that BPI protein product enhanced the early bactericidal
activity of sulfamethoxazole for all three organisms at 0-10 hours.
In FIG. 31, the growth curves for S. aureus with rBPI.sub.21 (open
squares), S. aureus without rPBI.sub.21 (filled squares) and S. pneumoniae
without rPBI.sub.21 (fried diamonds) overlap almost completely. FIG. 31
shows that BPI protein product enhanced the early bactericidal activity of
ciprofloxacin for S. pneumoniae and E. faecium at 0-10 hours.
In FIG. 32, the growth curves for S. pneumoniae with rPBI.sub.21 (open
diamonds) and S. pneumoniae without rPBI.sub.21 (filled diamonds) overlap
completely. FIG. 32 shows that BPI protein product enhanced the early
bactericidal effect of vancomycin for S. aureus and E. faecium at 0-10
hours.
The early time course of the bactericidal activity of BPI protein product
was studied for three clinical isolates (methicillin-resistant S. aureus,
S. pneumoniae, and E. faecium). Approximately 4 to 10 colonies of each
bacterial species, from 18 to 24 hour growth on either trypticase soy agar
plates (for S. aureus and E. faecium) or 5% sheep blood agar plates (for
S. pneumoniae) (Remel, Lenexa, Kans.), were emulsified in sterile water to
a density equivalent to an 0.5 McFarland standard; 100 .mu.L of this
bacterial inoculum was transferred to glass tubes containing 25 mL
Microscan.RTM. Pluronic Inoculum Water, such that the final concentration
of organisms was .about.4 to 7.times.10.sup.8 cells/mL, and rPBI.sub.21
was added to a final concentration of 16 .mu.g/mL. The tubes were mixed by
inversion, and a control and BPI sample were immediately removed and
diluted 1:100 in sterile water for colony counts. Similar samples were
taken at 7.5, 15, 30, 60, 90 and 120 minutes. The plates were incubated at
35.degree. C. to 37.degree. C. for 15 to 24 hours and the CFUs/mL were
measured by direct counts.
Some bactericidal activity was observed for all isolates afer 15 minutes of
incubation at room temperature. However, the strain considered susceptible
to the direct bactericidal effects of BPI protein product (S. pneumoniae)
was immediately reduced to 0 CFU at 7.5 minutes, while the killing curves
for "resistant" strains (S. aureus and E. faecium) were prolonged. There
was at least a three-log reduction in the number of organisms after the
BPI treatment; however, 0 CFU was never reached. CFU data is summarized in
Table 14 below.
TABLE 14
__________________________________________________________________________
Calculated CFUs/mL
0 mins
7.5 mins
15 mins
30 mins
60 mins
90 mins
120 mins
__________________________________________________________________________
S. pneumoniae control
179000
173000
153000
187000
169000
168000
143000
S. pneumoniae with rBPI.sub.21
6000
2 0 0 0 686 0
S. aurem control
488000
615000
440000
640000
480000
720000
410000
S. aureus with rBPI.sub.21
650000
5000 300 260 510 530 510
E. faecium control
276000
193000
265000
277000
265000
301000
212000
E. faecium with rBPI.sub.21
131000
129000
41000
3340
990 700 570
__________________________________________________________________________
To determine if the reason S. aureus organisms had survived the initial BPI
treatment was due to resistance to BPI protein product, colonies recovered
after 90 minutes of incubation with 16 .mu.g/mL rBPI.sub.21 were grown for
24 hours, prepared as previously described and incubated with an
additional 16 .mu.g/mL rBPI.sub.21. Control S. aureus that had not been
previously exposed to BPI protein product were also incubated with 16
.mu.g/mL rBPI.sub.21. After 30 minutes of incubation, the survivors and
controls were plated and counted. As seen in Table 15 below, the survivors
from an initial treatment with rBPI.sub.21 were not resistant to BPI
protein product.
TABLE 15
______________________________________
Control 16 .mu.g/mL rBPI.sub.21
______________________________________
S. aureus 5 .times. 10.sup.5 CFU/mL
3 .times. 10.sup.2 CFU/mL
S. aureus recovered after
4 .times. 10.sup.5 CFU/mL
2 .times. 10.sup.2 CFU/mL
90 min. incubation with
16 .mu.g/mL rBPI.sub.21
______________________________________
In another experiment, S. aureus organisms were incubated for 30 minutes in
16 .mu.g/mL rBPI.sub.21, and an aliquot was removed, plated and counted.
Half of the BPI-treated cell suspension was incubated with an additional
16 .mu.g/mL rBPI.sub.21. Incubation continued for both cell suspensions
for an additional 30 minutes. The additional rBPI.sub.21 treatment
decreased the relative number of survivors over the untreated control (84%
reduction/30 min. vs 34% control reduction/30 min.).
It should be noted that incubation in Mueller-Hinton broth inhibited the
complete killing of S. pneumoniae between 0 and 7.5 minutes. In additional
experiments conducted to explore this phenomenon, it was determined that
the addition of calcium chloride to the incubation medium appeared to
reduce the effectiveness of rBPI.sub.21 as measured by increased CFUs. A
preliminary experiment indicating that NaCl may also reduce the
effectiveness of rPBI.sub.21 (i.e., increased CFUs) suggests that
osmolality may be a factor.
EXAMPLE 20
IN VITRO EFFECT OF BPI PROTEIN PRODUCTS ALONE OR IN COMBINATION WITH
ANTIBIOTICS ON GRAM-POSITIVE ORGANISMS
BPI protein products are evaluated, using a multiwell antibiotic
susceptibility screening assay, for their effects alone or in combination
with penicillin, ampicillin or ciprofloxacin on a culture of Streptococcus
pneumoniae (especially penicillin-resistant organisms). BPI protein
products are also evaluated alone or in combination with the antibiotics
vancomycin, rifampin, ciprofloxacin, cefazolin, vancomycin/gentamicin and
ciprofloxacin/pip eracillin for their effects on the organism
Staphylococcus aureus (especially methicillin resistant organisms). BPI
protein products are evaluated alone or in combination with penicillin,
ampicillin, vancomycin, ciprofloxacin, penicillin/gentamicin, or
azithromycin for their effects on Enterococcus (multiply resistant
strains). The effect of BPI protein products alone or in combination with
vancomycin or clindamycin on Corynebacteria are evaluated.
EXAMPLE 21
IN VITRO ENHANCEMENT OF THE BACTERICIDAL ACTIVITY OF BPI PROTEIN PRODUCTS
BY SELECTED POLOXAMERS
The anti-bacterial activity of therapeutic compositions comprising a BPI
protein product and a variety of different poloxamer surfactants was
evaluated as described in co-owned, co-pending U.S. patent application
Ser. No. No. 08/372, 104, filed concurrently herewith the disclosure of
which is herein incorporated by reference. Briefly, therapeutic
compositions comprising a BPI protein product and a poloxamer surfactant
at concentrations ranging from 0.005 to 0.1% (weight/volume) were prepared
and incubated at 37.degree. C. with gram-positive organisms in water,
broth, or varying concentrations of serum. After incubation, the colony
forming units (CFU) of bacteria remaining were determined to ascertain
enhancement of anti-bacterial activity. Studies were also performed using
poloxamer surfactants that had been heat-treated using the following
procedure: (1) making a solution of the poloxamer in deionized water, (2)
heating the solution to a boil, (3) removing it from heat, (4) allowing it
to cool to room temperature, and (5) stirring until the poloxamer is
completely solubilized. Alternatively, in the heating step (2), the
solution may be boiled for up to 30 minutes or more.
It was observed that poloxamer 333 (heat-treated or not) and poloxamer 403
(heat-treated or not) and poloxamer 335 and heat-treated poloxamer 334
provided enhancement of the anti-bacterial activity of BPI protein
products against bacteria.
EXAMPLE 22
IN VIVO EFFECTS OF BPI PROTEIN PRODUCTS ALONE OR WITH ANTIBIOTICS
Preliminary experiments, in which BPI protein products alone or with
penicillin were administered to mice challenged intravenously with
5.times.10.sup.8 CFU of the S. aureus bacterial form of Example 3 above,
did not show an effect on mortality. This outcome was not unexpected in
view of the lack of effect of BPI protein products on that bacterial form
in the in vitro assays of Examples 2, 3 and 16. No effect on mortality was
observed in preliminary experiments in which mice were challenged with
1.times.10.sup.5 CFU of a S. pneumoniae bacterial form which appeared in a
preliminary experiment to be susceptible to rPBI.sub.21 alone in the
Microscan.RTM. system. Additional in vivo experiments will be performed in
a variety of other animal models, including using a variety of different
BPI protein products alone and in conjunction with a variety of different
antibiotics.
A. EVALUATION IN THE MOUSE PNEUMONIA MODEL
The effect on gram-positive infections of BPI protein products administered
alone or in combination with antibiotics is evaluated in a mouse pneumonia
model. Pneumonia is induced in mice by intranasal instillation of 3 drops
of an undiluted overnight culture of S. pneumoniae in trypticase soy broth
containing 5% goat serum. The challenge dose is administered under light
Metofane anesthesia with a 1-ml tuberculin syringe and a 22-gauge needle.
Infected mice are treated 24 hours after infection with a single
intravenous dose of BPI protein product alone, antibiotic alone, or the
combination of both agents. The mice are sacrificed 48 hours after
infection. The lungs are removed aseptically and cultured for the presence
of organism by touching a cut surface of the lung to a blood agar plate.
Under these conditions, at 48 hours after infection, untreated controls
exhibit consolidation of the lungs and heavily positive cultures. Further,
in this model, most of the untreated controls die within 96 hours after
infection.
B. EVALUATION IN THE RABBIT ENDOCARDITIS MODEL
The effect on gram-positive infections of BPI protein products administered
alone or in combination with antibiotics is evaluated in a rabbit
endocarditis model. Young New Zealand white rabbits weighing 1 to 2 kg are
anesthetized by the intravenous injection of 40 to 60 mg of
pentobarbitone. An incision is made parallel to the trachea, approximately
4 cm long and 1 cm to the right of the midline. To produce right-sided
endocarditis, the jugular vein is exposed and opened between ligatures.
The lower ligature is loosened, and a polyethylene catheter of 0.8 mm
external diameter and 0.4 mm internal diameter containing sterile saline
is passed toward the heart until resistance and pulsation indicates that
it has entered the right atrium or ventricle. The catheter is then secured
in place by tightening both ligatures, any excess is cut off, and the
distal end is sealed with a heated spatula before the skin is closed over
the catheter with silk sutures. For left-sided endocarditis, the right
carotid artery can be exposed through a similar incision, opened between
ligatures, and a catheter passed toward the heart until pulsation
resistance and reflux of arterial blood indicates that it has reached the
aortic valve or passed beyond into the left ventricle. It is then secured
as described above.
The presence of a catheter in the heart results in the development of
sterile vegetations consisting of small, rough whitish nodules 1 to 2 mm
in size, usually at points of contact between the catheter and the
endocardium. The sterile vegetations are infected by a single injection of
bacteria into an ear vein. If Streptococcus viridans is used for
infection, the infecting inoculum can be prepared by diluting an overnight
glucose broth culture and injecting approximately 10.sup.8 organisms in a
volume of 1 ml. Other gram-positive organisms can be used and may include
Micrococcus albus, S. aureus, S. epidermidis, and other strains. An
infecting inoculum can be prepared for these organisms by preparing a 1:10
dilution of an overnight glucose broth culture and injecting 1 mL
intravenously. BPI protein product alone, antibiotic alone, or the
combination treatment is given intravenously in daily doses. After days,
the rabbits are sacrificed and any vegetations found are removed
aseptically. The vegetations are weighed and homogenized in glucose broth.
Bacterial counts are determined by preparing serial dilutions in glucose
broth and incorporating the dilutions into blood agar pour plates. The CFU
of organism per g of wet weight is then calculated.
C. EVALUATION IN THE MOUSE INTRAPERITONEAL ABSCESS MODEL
The effect on gram-positive infections of BPI protein products administered
alone or in combination with antibiotics is evaluated in a mouse
intrapefitoneal abscess model. S. aureus is grown for 24 hours in
trypticase soy broth with constant shaking. Cultures are continuously
gassed with 100% oxygen to minimize .alpha.-hemolysin production. The
organisms are harvested by centrifugation, washed in saline contaning 1%
trypticase soy broth (v/v), and resuspended in the same diluent to
approximately 1.times.10.sup.10 to 5.times.10.sup.10 bacteria per ml.
Bacterial counts can be confirmed by plate counts.
Groups of female white Swiss mice weighing 20 to 30 g are inoculated
intraperitoneally with 0.5 ml of a suspension contaning 1 to
2.times.10.sup.9 bacteria. Beginning at 3 hours and at various intervals
thereafter, subgroups of infected animals selected at random are
sacrificed, and the clumped organisms or abscesses are aseptically removed
from the peritoneal cavities. All clumps or abscesses from a single animal
are ground and homogenized in 5 ml of saline containing 1% trypticase soy
broth. The suspension is serially diluted and the bacterial population
determined by plate counts.
D. EVALUATION IN THE MOUSE THIGH ABSCESS MODEL
The effect on gram-positive infections of BPI protein products administered
alone or in combination with antibiotics is evaluated in a mouse thigh
abscess model.
The thigh lesion model provides a nonlethal experimental infection to
evaluate the effectiveness of an antimicrobial and allows the measurement
of drug-pathogen interaction and drug pharmacokinetics in the infected
host. If the animals are made neutropenic, then the thigh model becomes an
excellent system for measuring the drug-microorganism interaction with
most of the host defense system eliminated.
Swiss outbred mice, preferably ICR mice, weighing 23 to 27 g are used. The
mice are made neutropenic by administering cyclophosphamide (150 and 100
mg/kg i.p.) on days 0 and 3, respectively. By day 4 severe neutropenia is
induced (<100 neutrophils/mm.sup.3) which lasts for two to three days. The
infecting organism is grown in broth to log phase and adjusted to an OD of
0.30 at 580 nm (.about.10.sup.6 to 10.sup.7 CFU/ml). The mice are infected
on day 4 by injecting 0.1 ml of this inoculum into each thigh while the
animals are under fight ether anesthesia. The infection is allowed to
proceed for 2 hours. The antimicrobial is then administered in graded
doses subcutaneously to groups of infected mice. Infected, untreated
animals serve as controls. Two to four mice from each treatment group are
sacrificed every hour for the first 4 hours and every 2 to 4 hours until
16 hours after treatment. Infected, untreated animals are killed at
similar times.
The thigh muscles are removed at each sampling period and homogenized
immediately in 9 ml of 0.85% NaCl with a Polytron tissue homogenizer.
Viable counts are determined after plating duplicate 10.mu. samples of
serial 10-fold dilutions of the homogenates on appropriate media. The log
CFU/thigh is determined at each time point for groups of treated and
untreated animals.
E. EVALUATION IN A RABBIT INTRAOCULAR INFECTION MODEL
The effect on gram-positive infections of BPI protein products administered
alone or in combination with antibiotics is evaluated in the following
model. The rabbits' eyes, anesthetized locally with 0.5% tetracaine
hydrochloride, are sterilized with Metaphen (1:4000) and flushed with
normal saline. Using a 26-gauge needle, approximately 0.2 ml containing
300,000 or 5,000 CFU or 0.02 ml containing 700 CFU of S. aureus is
inoculated into the center of the rabbit cornea, the anterior chamber, or
the vitreous of the eye. Cultures are taken at 24, 48, and 72 hours from
the corneas and anterior chambers of the rabbits' eyes that received
corneal inoculations. All corneas are flushed with Metaphen (1:4000) and
saline before culturing to eliminate surface contaminants.
Cultures are taken at the same intervals from the anterior chamber and
vitreous of rabbit eyes that were inoculated in the anterior chamber.
Vitreous humor is removed with a 19-gauge needle. The irises are also
cultured.
The eyes that are inoculated intravitreally are also cultured at 24, 48,
and 72 hours. Samples of the vitreous humor and anterior chamber as well
as the retina are used to determine the progress of the infection.
When 300,000 CFU in 0.2 ml of broth is inoculated into the corneas,
anterior chamber, and vitreous of the rabbit's eyes, a virulent
panophthalmitis is generally produced within 24 to 48 hours, and
destruction of the eye occurs within 72 hours regardless of the site of
inoculation. When 5000 CFU in 0.2 ml is inoculated into the corneas,
anterior chambers, and vitreous, a panophthalmitis generally results in 72
hours, with infections being most severe following intravitreal
inoculations and less intense when the anterior chamber is the site of
inoculation. When 700 CFU in 0.02 ml is used as the inoculum, the
infections are generally eliminated in the corneas and anterior chambers
within 24 hours but not in the vitreous.
Other models of ocular infection and disease known in the art [see, e.g.,
Sugar et al., Arch. Opthalmol., 104: 1230-1232 (1986) and Moon et al.,
Investigative Ophthalmol. Visual Science, 29: 1277-1284 (1988)] may be
used for the assessment of BPI protein product effects on ocular
conditions associated with bacterial infection, when the BPI protein
product is administered alone or in conjunction with antibiotics.
Numerous modifications and variations in the practice of the invention are
expected to occur to those skilled in the art upon consideration of the
foregoing description of the presently preferred embodiments thereof.
Consequently, the only limitations which should be placed upon the scope
of the present invention are those which appear in the appended claims.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 237
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "Domain I"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AlaSerGlnGlnGlyThrAlaAlaLeuGlnLysGluLeuLysArgIle
151015
LysIleProAspTyrSerAspSerPheLysIleLysHis
2025
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.14"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
GlyThrAlaAlaLeuGlnLysGluLeuLysArgIleLysIleProAsp
151015
TyrSerAspSerPheLysIleLysHisLeuGlyLysGlyHis
202530
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.4"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
LeuGlnLysGluLeuLysArgIleLysIleProAspTyrSerAspSer
151015
PheLysIleLysHisLeu
20
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.1"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GlnGlnGlyThrAlaAlaLeuGlnLysGluLeuLysArgIleLys
151015
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.54"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GlyThrAlaAlaLeuGlnLysGluLeuLysArgIleLysIlePro
151015
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "Domain II"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
SerSerGlnIleSerMetValProAsnValGlyLeuLysPheSerIle
151015
SerAsnAlaAsnIleLysIleSerGlyLysTrpLysAlaGlnLysArg
202530
PheLeuLys
35
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.2"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
IleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.8"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
LysTrpLysAlaGlnLysArgPheLeuLys
1510
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.58"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CysIleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.65 oxidized"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
CysIleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeuLys
151015
Cys
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.3"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
AsnValGlyLeuLysPheSerIleSerAsnAlaAsnIleLysIleSer
151015
GlyLysTrpLysAlaGlnLysArgPheLeuLys
2025
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "Domain III"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
ValHisValHisIleSerLysSerLysValGlyTrpLeuIleGlnLeu
151015
PheHisLysLysIleGluSerAlaLeuArgAsnLys
2025
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.11"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
LysSerLysValTrpLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.12"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
SerValHisValHisIleSerLysSerLysValGlyTrpLeuIleGln
151015
LeuPheHisLysLysIleGluSerAlaLeuArgAsnLys
2025
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.13"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.15"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
AlaLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.16"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
IleAlaIleSerGlyLysTrpLysAlaGlnLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.17"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
IleLysAlaSerGlyLysTrpLysAlaGlnLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.18"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
IleLysIleAlaGlyLysTrpLysAlaGlnLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.19"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
IleLysIleSerAlaLysTrpLysAlaGlnLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.20"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
IleLysIleSerGlyAlaTrpLysAlaGlnLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.21"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
IleLysIleSerGlyLysAlaLysAlaGlnLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.22"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
IleLysIleSerGlyLysTrpAlaAlaGlnLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.23"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
IleLysIleSerGlyLysTrpLysAlaAlaLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.24"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
IleLysIleSerGlyLysTrpLysAlaGlnAlaArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.25"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
IleLysIleSerGlyLysTrpLysAlaGlnLysAlaPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.26"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
IleLysIleSerGlyLysTrpLysAlaGlnLysArgAlaLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.27"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
IleLysIleSerGlyLysTrpLysAlaGlnLysArgPheAlaLys
151015
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.28"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
IleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeuAla
151015
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.59"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
IleLysIleSerGlyAlaTrpAlaAlaGlnLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.45"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
IleLysIleSerGlyLysTrpLysAlaAlaAlaArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.60"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
IleAlaIleSerGlyLysTrpLysAlaGlnLysArgPheLeuAla
151015
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.31"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
AlaSerLysValGlyTrpLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.32"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
LysAlaLysValGlyTrpLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.33"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
LysSerAlaValGlyTrpLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.34"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
LysSerLysAlaGlyTrpLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.35"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
LysSerLysValAlaTrpLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.36"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
LysSerLysValGlyAlaLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.37"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
LysSerLysValGlyTrpAlaIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.38"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
LysSerLysValGlyTrpLeuAlaGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.39"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
LysSerLysValGlyTrpLeuIleAlaLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.40"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
LysSerLysValGlyTrpLeuIleGlnAlaPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.41"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
LysSerLysValGlyTrpLeuIleGlnLeuAlaHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.42"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
LysSerLysValGlyTrpLeuIleGlnLeuPheAlaLysLys
1510
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.43"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
LysSerLysValGlyTrpLeuIleGlnLeuPheHisAlaLys
1510
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.44"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysAla
1510
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.56"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
IleLysIleSerGlyLysTrpLysAlaLysGlnArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.61"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
IleLysIleSerGlyLysPheLysAlaGlnLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.66"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 7
(D) OTHER INFORMATION: /label=D- Trp
/note="The amino acid at position 7 is
D- tryptophan"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
IleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.67"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6..8
(D) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 7 is
beta-1- naphthyl-substituted"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
IleLysIleSerGlyLysAlaLysAlaGlnLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.9"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
LysArgPheLeuLysLysTrpLysAlaGlnLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.30"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
LysTrpLysAlaGlnLysArgPheLeuLysLysSerLysValGlyTrp
151015
LeuIleGlnLeuPheHisLysLys
20
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.63"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
IleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeuLysLys
151015
SerLysValGlyTrpLeuIleGlnLeuPheHisLysLys
2025
(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.7"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
LysTrpLysAlaGlnLysArgPheLeuLysLysTrpLysAlaGlnLys
151015
ArgPheLeuLys
20
(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.10.1"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
LysArgPheLeuLysLysTrpLysAlaGlnLysArgPheLeuLysLys
151015
TrpLysAlaGlnLysArgPheLeuLys
2025
(2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.29"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLysLysSer
151015
LysValGlyTrpLeuIleGlnLeuPheHisLysLys
2025
(2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.46"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
LysTrpLysAlaAlaAlaArgPheLeuLysLysTrpLysAlaGlnLys
151015
ArgPheLeuLys
20
(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.47"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
LysTrpLysAlaGlnLysArgPheLeuLysLysTrpLysAlaAlaAla
151015
ArgPheLeuLys
20
(2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.48"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
LysTrpLysAlaAlaAlaArgPheLeuLysLysTrpLysAlaAlaAla
151015
ArgPheLeuLys
20
(2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.69"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
LysTrpLysAlaAlaAlaArgPheLeuLysLysTrpLysAlaAlaAla
151015
ArgPheLeuLysLysTrpLysAlaAlaAlaArgPheLeuLys
202530
(2) INFORMATION FOR SEQ ID NO:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.55"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:
GlyTrpLeuIleGlnLeuPheHisLysLysIleGluSerAlaLeuArg
151015
AsnLysMetAsnSer
20
(2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.73"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
IleLysIleSerGlyLysTrpLysAlaGlnPheArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.70"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 8..10
(D) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 7 is
beta-3- pyridyl-substituted"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
IleLysIleSerGlyLysAlaLysAlaGlnLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.71"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 13..15
(D) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 13 is
beta-3- pyridyl-substituted"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
IleLysIleSerGlyLysTrpLysAlaGlnLysArgAlaLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.10.2"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
GlnLysArgPheLeuLysLysTrpLysAlaGlnLysArgPheLeuLys
151015
LysTrpLysAlaGlnLysArgPheLeuLys
2025
(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.72"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1..3
(D) OTHER INFORMATION: /label=D- alanine
/note="The position 1 and position 2 alanine
residues are both D-alanine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:
AlaAlaIleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeu
151015
Lys
(2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.5"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
ValHisValHisIleSerLysSerLysValGlyTrpLeuIleGlnLeu
151015
PheHisLysLysIleGlu
20
(2) INFORMATION FOR SEQ ID NO:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.65 reduced"
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 1..17
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:
CysIleLysIleSerGlyLysTrpLysAlaGlnLysArgPheLeuLys
151015
Cys
(2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 487 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "rBPI"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:
MetArgGluAsnMetAlaArgGlyProCysAsnAlaProArgTrpVal
31-30-25-20
SerLeuMetValLeuValAlaIleGlyThrAlaValThrAlaAlaVal
15-10-51
AsnProGlyValValValArgIleSerGlnLysGlyLeuAspTyrAla
51015
SerGlnGlnGlyThrAlaAlaLeuGlnLysGluLeuLysArgIleLys
202530
IleProAspTyrSerAspSerPheLysIleLysHisLeuGlyLysGly
354045
HisTyrSerPheTyrSerMetAspIleArgGluPheGlnLeuProSer
50556065
SerGlnIleSerMetValProAsnValGlyLeuLysPheSerIleSer
707580
AsnAlaAsnIleLysIleSerGlyLysTrpLysAlaGlnLysArgPhe
859095
LeuLysMetSerGlyAsnPheAspLeuSerIleGluGlyMetSerIle
100105110
SerAlaAspLeuLysLeuGlySerAsnProThrSerGlyLysProThr
115120125
IleThrCysSerSerCysSerSerHisIleAsnSerValHisValHis
130135140145
IleSerLysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLys
150155160
IleGluSerAlaLeuArgAsnLysMetAsnSerGlnValCysGluLys
165170175
ValThrAsnSerValSerSerLysLeuGlnProTyrPheGlnThrLeu
180185190
ProValMetThrLysIleAspSerValAlaGlyIleAsnTyrGlyLeu
195200205
ValAlaProProAlaThrThrAlaGluThrLeuAspValGlnMetLys
210215220225
GlyGluPheTyrSerGluAsnHisHisAsnProProProPheAlaPro
230235240
ProValMetGluPheProAlaAlaHisAspArgMetValTyrLeuGly
245250255
LeuSerAspTyrPhePheAsnThrAlaGlyLeuValTyrGlnGluAla
260265270
GlyValLeuLysMetThrLeuArgAspAspMetIleProLysGluSer
275280285
LysPheArgLeuThrThrLysPhePheGlyThrPheLeuProGluVal
290295300305
AlaLysLysPheProAsnMetLysIleGlnIleHisValSerAlaSer
310315320
ThrProProHisLeuSerValGlnProThrGlyLeuThrPheTyrPro
325330335
AlaValAspValGlnAlaPheAlaValLeuProAsnSerSerLeuAla
340345350
SerLeuPheLeuIleGlyMetHisThrThrGlySerMetGluValSer
355360365
AlaGluSerAsnArgLeuValGlyGluLeuLysLeuAspArgLeuLeu
370375380385
LeuGluLeuLysHisSerAsnIleGlyProPheProValGluLeuLeu
390395400
GlnAspIleMetAsnTyrIleValProIleLeuValLeuProArgVal
405410415
AsnGluLysLeuGlnLysGlyPheProLeuProThrProAlaArgVal
420425430
GlnLeuTyrAsnValValLeuGlnProHisGlnAsnPheLeuLeuPhe
435440445
GlyAlaAspValValTyrLys
450455
(2) INFORMATION FOR SEQ ID NO:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.74"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLysLysTrp
151015
LysAlaGlnLysArgPheLeuLys
20
(2) INFORMATION FOR SEQ ID NO:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.76"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10..12
(D) OTHER INFORMATION: /label=D- Phe
/note="The amino acid at position 11 is
D- phenylalanine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
IleLysIleSerGlyLysTrpLysAlaGlnPheArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.77"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
IleLysIleSerGlyLysTrpLysAlaGlnTrpArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.79"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
IleLysIleSerGlyLysTrpLysAlaLysLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.80"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10..12
(D) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 11 is
beta-1- naphthyl-substituted"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
IleLysIleSerGlyLysTrpLysAlaGlnAlaArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.81"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:
IleLysIleSerGlyLysTrpLysAlaPheLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.82"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:
LysSerLysValGlyTrpLeuIleGlnLeuTrpHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.83"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10..12
(D) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 6 is
beta-1- naphthyl-substituted"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:
LysSerLysValGlyAlaLysIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.84"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6..8
(D) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 7 is
beta-1- naphthyl-substituted"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:
IleLysIleSerGlyLysAlaLysAlaGlnPheArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.85"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:
LysSerLysValLeuTrpLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.86"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:
LysSerLysValGlyTrpLeuIleLeuLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.87"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:
LysSerLysValGlyTrpLeuIleGlnLeuPheLeuLysLys
1510
(2) INFORMATION FOR SEQ ID NO:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.88"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:
IleLysIleSerGlyLysTrpLysAlaPhePheArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.98"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(D) OTHER INFORMATION: /label=Substituted-Trp
/note="The alanine at position 2 is
beta-1- naphthyl-substituted"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:
LysTrpLysAlaGlnPheArgPheLeuLysLysSerLysValGlyTrp
151015
LeuIlePheLeuPheHisLysLys
20
(2) INFORMATION FOR SEQ ID NO:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.89"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6..8
(D) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 7 is
beta-1- naphthyl-substituted"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:
IleLysIleSerGlyLysAlaLysAlaPheLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.90"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6..8
(D) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 7 is
beta-1- naphthyl-substituted"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:
IleLysIleSerGlyLysAlaLysAlaPhePheArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.91"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:
LysSerLysValGlyTrpLeuIlePheLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.92"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:
LysSerLysValGlyTrpLeuIleLysLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.93"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6..8
(D) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 7 is
beta-1- naphthyl-substituted"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:
IleLysIleSerGlyLysAlaLysAlaGlnPheArgPheLeuLysLys
151015
SerLysValGlyTrpLeuIleGlnLeuPheHisLysLys
2025
(2) INFORMATION FOR SEQ ID NO:89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.94"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:
LysSerLysValGlyTrpLeuIleGlnLeuPhePheLysLys
1510
(2) INFORMATION FOR SEQ ID NO:90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.95"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:
LysSerLysValPheTrpLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.96"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:
LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysPhe
1510
(2) INFORMATION FOR SEQ ID NO:92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.97"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:
LysSerLysValLysTrpLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.99"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:
LysTrpLysAlaGlnTrpArgPheLeuLysLysTrpLysAlaGlnTrp
151015
ArgPheLeuLysLysTrpLysAlaGlnTrpArgPheLeuLys
202530
(2) INFORMATION FOR SEQ ID NO:94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.100"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:
LysSerLysValLysTrpLeuIleLysLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.101"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:
LysSerLysValLysTrpLeuIleLysLeuPhePheLysPheLysSer
151015
LysValLysTrpLeuIleLysLeuPhePheLysPhe
2025
(2) INFORMATION FOR SEQ ID NO:96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.102"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:
LysTrpLysAlaGlnPheArgPheLeuLysLysSerLysValGlyTrp
151015
LeuIleLeuLeuPheHisLysLys
20
(2) INFORMATION FOR SEQ ID NO:97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1443 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..1443
(ix) FEATURE:
(A) NAME/KEY: mat.sub.-- peptide
(B) LOCATION: 76..1443
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "rLBP"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:
ATGGGGGCCTTGGCCAGAGCCCTGCCGTCCATACTGCTGGCATTGCTG48
MetGlyAlaLeuAlaArgAlaLeuProSerIleLeuLeuAlaLeuLeu
25-20-15-10
CTTACGTCCACCCCAGAGGCTCTGGGTGCCAACCCCGGCTTGGTCGCC96
LeuThrSerThrProGluAlaLeuGlyAlaAsnProGlyLeuValAla
515
AGGATCACCGACAAGGGACTGCAGTATGCGGCCCAGGAGGGGCTATTG144
ArgIleThrAspLysGlyLeuGlnTyrAlaAlaGlnGluGlyLeuLeu
101520
GCTCTGCAGAGTGAGCTGCTCAGGATCACGCTGCCTGACTTCACCGGG192
AlaLeuGlnSerGluLeuLeuArgIleThrLeuProAspPheThrGly
253035
GACTTGAGGATCCCCCACGTCGGCCGTGGGCGCTATGAGTTCCACAGC240
AspLeuArgIleProHisValGlyArgGlyArgTyrGluPheHisSer
40455055
CTGAACATCCACAGCTGTGAGCTGCTTCACTCTGCGCTGAGGCCTGTC288
LeuAsnIleHisSerCysGluLeuLeuHisSerAlaLeuArgProVal
606570
CCTGGCCAGGGCCTGAGTCTCAGCATCTCCGACTCCTCCATCCGGGTC336
ProGlyGlnGlyLeuSerLeuSerIleSerAspSerSerIleArgVal
758085
CAGGGCAGGTGGAAGGTGCGCAAGTCATTCTTCAAACTACAGGGCTCC384
GlnGlyArgTrpLysValArgLysSerPhePheLysLeuGlnGlySer
9095100
TTTGATGTCAGTGTCAAGGGCATCAGCATTTCGGTCAACCTCCTGTTG432
PheAspValSerValLysGlyIleSerIleSerValAsnLeuLeuLeu
105110115
GGCAGCGAGTCCTCCGGGAGGCCCACAGTTACTGCCTCCAGCTGCAGC480
GlySerGluSerSerGlyArgProThrValThrAlaSerSerCysSer
120125130135
AGTGACATCGCTGACGTGGAGGTGGACATGTCGGGAGACTTGGGGTGG528
SerAspIleAlaAspValGluValAspMetSerGlyAspLeuGlyTrp
140145150
CTGTTGAACCTCTTCCACAACCAGATTGAGTCCAAGTTCCAGAAAGTA576
LeuLeuAsnLeuPheHisAsnGlnIleGluSerLysPheGlnLysVal
155160165
CTGGAGAGCAGGATTTGCGAAATGATCCAGAAATCGGTGTCCTCCGAT624
LeuGluSerArgIleCysGluMetIleGlnLysSerValSerSerAsp
170175180
CTACAGCCTTATCTCCAAACTCTGCCAGTTACAACAGAGATTGACAGT672
LeuGlnProTyrLeuGlnThrLeuProValThrThrGluIleAspSer
185190195
TTCGCCGACATTGATTATAGCTTAGTGGAAGCCCCTCGGGCAACAGCC720
PheAlaAspIleAspTyrSerLeuValGluAlaProArgAlaThrAla
200205210215
CAGATGCTGGAGGTGATGTTTAAGGGTGAAATCTTTCATCGTAACCAC768
GlnMetLeuGluValMetPheLysGlyGluIlePheHisArgAsnHis
220225230
CGTTCTCCAGTTACCCTCCTTGCTGCAGTCATGAGCCTTCCTGAGGAA816
ArgSerProValThrLeuLeuAlaAlaValMetSerLeuProGluGlu
235240245
CACAACAAAATGGTCTACTTTGCCATCTCGGATTATGTCTTCAACACG864
HisAsnLysMetValTyrPheAlaIleSerAspTyrValPheAsnThr
250255260
GCCAGCCTGGTTTATCATGAGGAAGGATATCTGAACTTCTCCATCACA912
AlaSerLeuValTyrHisGluGluGlyTyrLeuAsnPheSerIleThr
265270275
GATGAGATGATACCGCCTGACTCTAATATCCGACTGACCACCAAGTCC960
AspGluMetIleProProAspSerAsnIleArgLeuThrThrLysSer
280285290295
TTCCGACCCTTCGTCCCACGGTTAGCCAGGCTCTACCCCAACATGAAC1008
PheArgProPheValProArgLeuAlaArgLeuTyrProAsnMetAsn
300305310
CTGGAACTCCAGGGATCAGTGCCCTCTGCTCCGCTCCTGAACTTCAGC1056
LeuGluLeuGlnGlySerValProSerAlaProLeuLeuAsnPheSer
315320325
CCTGGGAATCTGTCTGTGGACCCCTATATGGAGATAGATGCCTTTGTG1104
ProGlyAsnLeuSerValAspProTyrMetGluIleAspAlaPheVal
330335340
CTCCTGCCCAGCTCCAGCAAGGAGCCTGTCTTCCGGCTCAGTGTGGCC1152
LeuLeuProSerSerSerLysGluProValPheArgLeuSerValAla
345350355
ACTAATGTGTCCGCCACCTTGACCTTCAATACCAGCAAGATCACTGGG1200
ThrAsnValSerAlaThrLeuThrPheAsnThrSerLysIleThrGly
360365370375
TTCCTGAAGCCAGGAAAGGTAAAAGTGGAACTGAAAGAATCCAAAGTT1248
PheLeuLysProGlyLysValLysValGluLeuLysGluSerLysVal
380385390
GGACTATTCAATGCAGAGCTGTTGGAAGCGCTCCTCAACTATTACATC1296
GlyLeuPheAsnAlaGluLeuLeuGluAlaLeuLeuAsnTyrTyrIle
395400405
CTTAACACCTTCTACCCCAAGTTCAATGATAAGTTGGCCGAAGGCTTC1344
LeuAsnThrPheTyrProLysPheAsnAspLysLeuAlaGluGlyPhe
410415420
CCCCTTCCTCTGCTGAAGCGTGTTCAGCTCTACGACCTTGGGCTGCAG1392
ProLeuProLeuLeuLysArgValGlnLeuTyrAspLeuGlyLeuGln
425430435
ATCCATAAGGACTTCCTGTTCTTGGGTGCCAATGTCCAATACATGAGA1440
IleHisLysAspPheLeuPheLeuGlyAlaAsnValGlnTyrMetArg
440445450455
GTT1443
Val
(2) INFORMATION FOR SEQ ID NO:98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 481 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "rLBP"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:
MetGlyAlaLeuAlaArgAlaLeuProSerIleLeuLeuAlaLeuLeu
25-20-15-10
LeuThrSerThrProGluAlaLeuGlyAlaAsnProGlyLeuValAla
515
ArgIleThrAspLysGlyLeuGlnTyrAlaAlaGlnGluGlyLeuLeu
101520
AlaLeuGlnSerGluLeuLeuArgIleThrLeuProAspPheThrGly
253035
AspLeuArgIleProHisValGlyArgGlyArgTyrGluPheHisSer
40455055
LeuAsnIleHisSerCysGluLeuLeuHisSerAlaLeuArgProVal
606570
ProGlyGlnGlyLeuSerLeuSerIleSerAspSerSerIleArgVal
758085
GlnGlyArgTrpLysValArgLysSerPhePheLysLeuGlnGlySer
9095100
PheAspValSerValLysGlyIleSerIleSerValAsnLeuLeuLeu
105110115
GlySerGluSerSerGlyArgProThrValThrAlaSerSerCysSer
120125130135
SerAspIleAlaAspValGluValAspMetSerGlyAspLeuGlyTrp
140145150
LeuLeuAsnLeuPheHisAsnGlnIleGluSerLysPheGlnLysVal
155160165
LeuGluSerArgIleCysGluMetIleGlnLysSerValSerSerAsp
170175180
LeuGlnProTyrLeuGlnThrLeuProValThrThrGluIleAspSer
185190195
PheAlaAspIleAspTyrSerLeuValGluAlaProArgAlaThrAla
200205210215
GlnMetLeuGluValMetPheLysGlyGluIlePheHisArgAsnHis
220225230
ArgSerProValThrLeuLeuAlaAlaValMetSerLeuProGluGlu
235240245
HisAsnLysMetValTyrPheAlaIleSerAspTyrValPheAsnThr
250255260
AlaSerLeuValTyrHisGluGluGlyTyrLeuAsnPheSerIleThr
265270275
AspGluMetIleProProAspSerAsnIleArgLeuThrThrLysSer
280285290295
PheArgProPheValProArgLeuAlaArgLeuTyrProAsnMetAsn
300305310
LeuGluLeuGlnGlySerValProSerAlaProLeuLeuAsnPheSer
315320325
ProGlyAsnLeuSerValAspProTyrMetGluIleAspAlaPheVal
330335340
LeuLeuProSerSerSerLysGluProValPheArgLeuSerValAla
345350355
ThrAsnValSerAlaThrLeuThrPheAsnThrSerLysIleThrGly
360365370375
PheLeuLysProGlyLysValLysValGluLeuLysGluSerLysVal
380385390
GlyLeuPheAsnAlaGluLeuLeuGluAlaLeuLeuAsnTyrTyrIle
395400405
LeuAsnThrPheTyrProLysPheAsnAspLysLeuAlaGluGlyPhe
410415420
ProLeuProLeuLeuLysArgValGlnLeuTyrAspLeuGlyLeuGln
425430435
IleHisLysAspPheLeuPheLeuGlyAlaAsnValGlnTyrMetArg
440445450455
Val
(2) INFORMATION FOR SEQ ID NO:99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.57"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:
CysIleLysIleSerGlyLysTrpLysAlaGlnLysArgProLeuCys
151015
(2) INFORMATION FOR SEQ ID NO:100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.75"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:
IleLysLysArgAlaIleSerPheLeuGlyLysLysTrpGlnLys
151015
(2) INFORMATION FOR SEQ ID NO:101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.282"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:
LysTrpLysAlaPhePheArgPheLeuLysLysTrpLysAlaPhePhe
151015
ArgPheLeuLys
20
(2) INFORMATION FOR SEQ ID NO:102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.103"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:
IleLysIleSerGlyLysTrpLysAlaTrpLysArgPheLeuLysLys
151015
(2) INFORMATION FOR SEQ ID NO:103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.104"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:
LysSerLysValGlyTrpLeuIleSerLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.105"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 13
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 13 is beta-1-
naphthyl- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:
IleLysIleSerGlyLysTrpLysAlaTrpLysArgAlaLeuLysLys
151015
(2) INFORMATION FOR SEQ ID NO:105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.106"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:
LysSerLysValGlyTrpLeuIleThrLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.107"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:
LysSerLysValGlyTrpLeuIleGlnLeuPheTrpLysLys
1510
(2) INFORMATION FOR SEQ ID NO:107:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.108"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:
LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysTrp
1510
(2) INFORMATION FOR SEQ ID NO:108:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.109"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 11
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 11 is beta-1-
naphthyl- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:
LysSerLysValGlyTrpLeuIleGlnLeuAlaHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:109:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.110"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 12
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 12 is beta-1-
naphthyl- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:
LysSerLysValGlyTrpLeuIleGlnLeuPheAlaLysLys
1510
(2) INFORMATION FOR SEQ ID NO:110:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.111"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 14
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 14 is beta-1-
naphthyl- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:
LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysAla
1510
(2) INFORMATION FOR SEQ ID NO:111:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.112"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 7
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 7 is beta-1-
naphthyl- substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 11
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 11 is beta-1-
naphthyl- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:
IleLysIleSerGlyLysAlaLysAlaGlnAlaArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.113"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:
LysSerLysValGlyTrpLeuIleGlnPhePheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.114"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:
LysTrpGlnLeuArgSerLysGlyLysIleLysIlePheLysAla
151015
(2) INFORMATION FOR SEQ ID NO:114:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.116"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 6 is beta-1-
naphthyl- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:
LysSerLysValLysAlaLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.119"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 7
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 7 is beta-1-
naphthyl- substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 10 is beta-1-
naphthyl- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:
IleLysIleSerGlyLysAlaLysAlaAlaLysArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:116:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.120"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:
IleLysIleSerGlyLysTrpLysAlaGlnLysArgLysLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:117:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.121"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 10 is beta-1-
naphthyl- substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 11
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 11 is beta-1-
naphthyl- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:
IleLysIleSerGlyLysTrpLysAlaAlaAlaArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:118:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.122"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 7
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 7 is beta-1-
naphthyl- substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 10 is beta-1-
naphthyl- substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 11
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 11 is beta-1-
naphthyl- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:
IleLysIleSerGlyLysAlaLysAlaAlaAlaArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:119:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.123"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 9
(C) OTHER INFORMATION: /label=Substituted-Phe
/note="The phenylalanine at position 9 is
p-amino- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:
LysSerLysValGlyTrpLeuIlePheLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:120:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.124"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:
LysSerLysValLysTrpLeuIleGlnLeuTrpHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:121:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.125"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:
LysSerLysValGlyTrpLeuIleTyrLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:122:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.126"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=D- Trp
/note="The amino acid at position 6 is
D- tryptophan."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:
LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:123:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.127"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:
LysSerLysValGlyPheLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:124:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.128"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=D- Phe
/note="The amino acid at position 6 is
D- phenylalanine."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:
LysSerLysValGlyPheLeuIleGlnLeuProHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:125:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.129"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 6 is
D-1-beta-1- naphthyl-
substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:
LysSerLysValGlyAlaLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:126:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.130"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 6 is
2-beta-1- naphthyl-
substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:
LysSerLysValGlyAlaLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:127:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.131"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 6 is
D-2-beta-1- naphthyl-
substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:
LysSerLysValGlyAlaLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:128:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.132"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 6 is
pyridyl- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:
LysSerLysValGlyAlaLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:129:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.133"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Phe
/note="The phenylalanine at position 6 is
para-amino-
substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:129:
LysSerLysValGlyPheLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:130:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.134"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(C) OTHER INFORMATION: /label=Substituted-Phe
/note="The phenylalanine at position 5 is
para-amino- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:
LysSerLysValPheTrpLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:131:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.135"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:131:
LysSerLysValGlyLysLeuIleGlnLeuProHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:132:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.136"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:
IleLysIleSerGlyLysTrpLysAlaGlnGluArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:133:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.137"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:133:
CysLysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLysCys
151015
(2) INFORMATION FOR SEQ ID NO:134:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.138"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:
LysSerLysValLysPheLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:135:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.139"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:
LysSerLysValGlyTyrLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:136:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.140"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 1 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 2 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:
AlaAlaArgPheLeuLysPhe
15
(2) INFORMATION FOR SEQ ID NO:137:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.141"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:
IleLysIleSerGlyLysTrpLysAlaGlnLysArgTrpLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:138:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.142"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:138:
LysSerLysValGlyTrpLeuIleGlnTrpPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:139:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.143"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 10 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:139:
LysSerLysValGlyTrpLeuIleGlnAlaPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:140:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.144"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 6 is
cyclohexyl- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:140:
LysSerLysValGlyAlaLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:141:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.145"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:141:
LysTrpLysAlaAlaAlaArgPheLeuLysLysSerLysValGlyTrp
151015
LeuIleGlnLeuPheHisLysLys
20
(2) INFORMATION FOR SEQ ID NO:142:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.146"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 12
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 12 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 14
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 14 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:142:
LysSerLysValGlyTrpLeuIleGlnLeuPheAlaLysAla
1510
(2) INFORMATION FOR SEQ ID NO:143:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.147"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:143:
IleLysIleSerGlyLysTrpLysAlaGluLysLysPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:144:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.148"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 6 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 12
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 12 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:144:
LysSerLysValGlyAlaLeuIleGlnLeuPheAlaLysLys
1510
(2) INFORMATION FOR SEQ ID NO:145:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1813 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 31..1491
(ix) FEATURE:
(A) NAME/KEY: mat.sub.-- peptide
(B) LOCATION: 124..1491
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "rBPI"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:145:
CAGGCCTTGAGGTTTTGGCAGCTCTGGAGGATGAGAGAGAACATGGCCAGGGGC54
MetArgGluAsnMetAlaArgGly
31-30-25
CCTTGCAACGCGCCGAGATGGGTGTCCCTGATGGTGCTCGTCGCCATA102
ProCysAsnAlaProArgTrpValSerLeuMetValLeuValAlaIle
20-15- 10
GGCACCGCCGTGACAGCGGCCGTCAACCCTGGCGTCGTGGTCAGGATC150
GlyThrAlaValThrAlaAlaValAsnProGlyValValValArgIle
515
TCCCAGAAGGGCCTGGACTACGCCAGCCAGCAGGGGACGGCCGCTCTG198
SerGlnLysGlyLeuAspTyrAlaSerGlnGlnGlyThrAlaAlaLeu
10152025
CAGAAGGAGCTGAAGAGGATCAAGATTCCTGACTACTCAGACAGCTTT246
GlnLysGluLeuLysArgIleLysIleProAspTyrSerAspSerPhe
303540
AAGATCAAGCATCTTGGGAAGGGGCATTATAGCTTCTACAGCATGGAC294
LysIleLysHisLeuGlyLysGlyHisTyrSerPheTyrSerMetAsp
455055
ATCCGTGAATTCCAGCTTCCCAGTTCCCAGATAAGCATGGTGCCCAAT342
IleArgGluPheGlnLeuProSerSerGlnIleSerMetValProAsn
606570
GTGGGCCTTAAGTTCTCCATCAGCAACGCCAATATCAAGATCAGCGGG390
ValGlyLeuLysPheSerIleSerAsnAlaAsnIleLysIleSerGly
758085
AAATGGAAGGCACAAAAGAGATTCTTAAAAATGAGCGGCAATTTTGAC438
LysTrpLysAlaGlnLysArgPheLeuLysMetSerGlyAsnPheAsp
9095100105
CTGAGCATAGAAGGCATGTCCATTTCGGCTGATCTGAAGCTGGGCAGT486
LeuSerIleGluGlyMetSerIleSerAlaAspLeuLysLeuGlySer
110115120
AACCCCACGTCAGGCAAGCCCACCATCACCTGCTCCAGCTGCAGCAGC534
AsnProThrSerGlyLysProThrIleThrCysSerSerCysSerSer
125130135
CACATCAACAGTGTCCACGTGCACATCTCAAAGAGCAAAGTCGGGTGG582
HisIleAsnSerValHisValHisIleSerLysSerLysValGlyTrp
140145150
CTGATCCAACTCTTCCACAAAAAAATTGAGTCTGCGCTTCGAAACAAG630
LeuIleGlnLeuPheHisLysLysIleGluSerAlaLeuArgAsnLys
155160165
ATGAACAGCCAGGTCTGCGAGAAAGTGACCAATTCTGTATCCTCCAAG678
MetAsnSerGlnValCysGluLysValThrAsnSerValSerSerLys
170175180185
CTGCAACCTTATTTCCAGACTCTGCCAGTAATGACCAAAATAGATTCT726
LeuGlnProTyrPheGlnThrLeuProValMetThrLysIleAspSer
190195200
GTGGCTGGAATCAACTATGGTCTGGTGGCACCTCCAGCAACCACGGCT774
ValAlaGlyIleAsnTyrGlyLeuValAlaProProAlaThrThrAla
205210215
GAGACCCTGGATGTACAGATGAAGGGGGAGTTTTACAGTGAGAACCAC822
GluThrLeuAspValGlnMetLysGlyGluPheTyrSerGluAsnHis
220225230
CACAATCCACCTCCCTTTGCTCCACCAGTGATGGAGTTTCCCGCTGCC870
HisAsnProProProPheAlaProProValMetGluPheProAlaAla
235240245
CATGACCGCATGGTATACCTGGGCCTCTCAGACTACTTCTTCAACACA918
HisAspArgMetValTyrLeuGlyLeuSerAspTyrPhePheAsnThr
250255260265
GCCGGGCTTGTATACCAAGAGGCTGGGGTCTTGAAGATGACCCTTAGA966
AlaGlyLeuValTyrGlnGluAlaGlyValLeuLysMetThrLeuArg
270275280
GATGACATGATTCCAAAGGAGTCCAAATTTCGACTGACAACCAAGTTC1014
AspAspMetIleProLysGluSerLysPheArgLeuThrThrLysPhe
285290295
TTTGGAACCTTCCTACCTGAGGTGGCCAAGAAGTTTCCCAACATGAAG1062
PheGlyThrPheLeuProGluValAlaLysLysPheProAsnMetLys
300305310
ATACAGATCCATGTCTCAGCCTCCACCCCGCCACACCTGTCTGTGCAG1110
IleGlnIleHisValSerAlaSerThrProProHisLeuSerValGln
315320325
CCCACCGGCCTTACCTTCTACCCTGCCGTGGATGTCCAGGCCTTTGCC1158
ProThrGlyLeuThrPheTyrProAlaValAspValGlnAlaPheAla
330335340345
GTCCTCCCCAACTCCTCCCTGGCTTCCCTCTTCCTGATTGGCATGCAC1206
ValLeuProAsnSerSerLeuAlaSerLeuPheLeuIleGlyMetHis
350355360
ACAACTGGTTCCATGGAGGTCAGCGCCGAGTCCAACAGGCTTGTTGGA1254
ThrThrGlySerMetGluValSerAlaGluSerAsnArgLeuValGly
365370375
GAGCTCAAGCTGGATAGGCTGCTCCTGGAACTGAAGCACTCAAATATT1302
GluLeuLysLeuAspArgLeuLeuLeuGluLeuLysHisSerAsnIle
380385390
GGCCCCTTCCCGGTTGAATTGCTGCAGGATATCATGAACTACATTGTA1350
GlyProPheProValGluLeuLeuGlnAspIleMetAsnTyrIleVal
395400405
CCCATTCTTGTGCTGCCCAGGGTTAACGAGAAACTACAGAAAGGCTTC1398
ProIleLeuValLeuProArgValAsnGluLysLeuGlnLysGlyPhe
410415420425
CCTCTCCCGACGCCGGCCAGAGTCCAGCTCTACAACGTAGTGCTTCAG1446
ProLeuProThrProAlaArgValGlnLeuTyrAsnValValLeuGln
430435440
CCTCACCAGAACTTCCTGCTGTTCGGTGCAGACGTTGTCTATAAA1491
ProHisGlnAsnPheLeuLeuPheGlyAlaAspValValTyrLys
445450455
TGAAGGCACCAGGGGTGCCGGGGGCTGTCAGCCGCACCTGTTCCTGATGGGCTGTGGGGC1551
ACCGGCTGCCTTTCCCCAGGGAATCCTCTCCAGATCTTAACCAAGAGCCCCTTGCAAACT1611
TCTTCGACTCAGATTCAGAAATGATCTAAACACGAGGAAACATTATTCATTGGAAAAGTG1671
CATGGTGTGTATTTTAGGGATTATGAGCTTCTTTCAAGGGCTAAGGCTGCAGAGATATTT1731
CCTCCAGGAATCGTGTTTCAATTGTAACCAAGAAATTTCCATTTGTGCTTCATGAAAAAA1791
AACTTCTGGTTTTTTTCATGTG1813
(2) INFORMATION FOR SEQ ID NO:146:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 487 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:146:
MetArgGluAsnMetAlaArgGlyProCysAsnAlaProArgTrpVal
31-30-25-20
SerLeuMetValLeuValAlaIleGlyThrAlaValThrAlaAlaVal
15-10-51
AsnProGlyValValValArgIleSerGlnLysGlyLeuAspTyrAla
51015
SerGlnGlnGlyThrAlaAlaLeuGlnLysGluLeuLysArgIleLys
202530
IleProAspTyrSerAspSerPheLysIleLysHisLeuGlyLysGly
354045
HisTyrSerPheTyrSerMetAspIleArgGluPheGlnLeuProSer
50556065
SerGlnIleSerMetValProAsnValGlyLeuLysPheSerIleSer
707580
AsnAlaAsnIleLysIleSerGlyLysTrpLysAlaGlnLysArgPhe
859095
LeuLysMetSerGlyAsnPheAspLeuSerIleGluGlyMetSerIle
100105110
SerAlaAspLeuLysLeuGlySerAsnProThrSerGlyLysProThr
115120125
IleThrCysSerSerCysSerSerHisIleAsnSerValHisValHis
130135140145
IleSerLysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLys
150155160
IleGluSerAlaLeuArgAsnLysMetAsnSerGlnValCysGluLys
165170175
ValThrAsnSerValSerSerLysLeuGlnProTyrPheGlnThrLeu
180185190
ProValMetThrLysIleAspSerValAlaGlyIleAsnTyrGlyLeu
195200205
ValAlaProProAlaThrThrAlaGluThrLeuAspValGlnMetLys
210215220225
GlyGluPheTyrSerGluAsnHisHisAsnProProProPheAlaPro
230235240
ProValMetGluPheProAlaAlaHisAspArgMetValTyrLeuGly
245250255
LeuSerAspTyrPhePheAsnThrAlaGlyLeuValTyrGlnGluAla
260265270
GlyValLeuLysMetThrLeuArgAspAspMetIleProLysGluSer
275280285
LysPheArgLeuThrThrLysPhePheGlyThrPheLeuProGluVal
290295300305
AlaLysLysPheProAsnMetLysIleGlnIleHisValSerAlaSer
310315320
ThrProProHisLeuSerValGlnProThrGlyLeuThrPheTyrPro
325330335
AlaValAspValGlnAlaPheAlaValLeuProAsnSerSerLeuAla
340345350
SerLeuPheLeuIleGlyMetHisThrThrGlySerMetGluValSer
355360365
AlaGluSerAsnArgLeuValGlyGluLeuLysLeuAspArgLeuLeu
370375380385
LeuGluLeuLysHisSerAsnIleGlyProPheProValGluLeuLeu
390395400
GlnAspIleMetAsnTyrIleValProIleLeuValLeuProArgVal
405410415
AsnGluLysLeuGlnLysGlyPheProLeuProThrProAlaArgVal
420425430
GlnLeuTyrAsnValValLeuGlnProHisGlnAsnPheLeuLeuPhe
435440445
GlyAlaAspValValTyrLys
450455
(2) INFORMATION FOR SEQ ID NO:147:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.149"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:147:
LysTrpLysValPheLysLysIleGluLysLysSerLysValGlyTrp
151015
LeuIleGlnLeuPheHisLysLys
20
(2) INFORMATION FOR SEQ ID NO:148:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.150"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:148:
LysTrpAlaPheAlaLysLysGlnLysLysArgLeuLysArgGlnTrp
151015
LeuLysLysPhe
20
(2) INFORMATION FOR SEQ ID NO:149:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.153"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:149:
LysTrpLysAlaGlnLysArgPheLeuLysLysTrpLysAlaGlnLys
151015
ArgPheLeuLysLysTrpLysAlaGlnLysArgPheLeuLys
202530
(2) INFORMATION FOR SEQ ID NO:150:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.154"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 5 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 6 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:150:
LysTrpLysAlaAlaAlaArgPheLeuLysLysTrpLysAlaGlnLys
151015
ArgPheLeuLys
20
(2) INFORMATION FOR SEQ ID NO:151:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.155"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 15
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 15 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 16
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 16 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:151:
LysTrpLysAlaGlnLysArgPheLeuLysLysTrpLysAlaAlaAla
151015
ArgPheLeuLys
20
(2) INFORMATION FOR SEQ ID NO:152:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.156"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 5 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 6 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 15
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 15 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 16
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 16 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:152:
LysTrpLysAlaAlaAlaArgPheLeuLysLysTrpLysAlaAlaAla
151015
ArgPheLeuLys
20
(2) INFORMATION FOR SEQ ID NO:153:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.157"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 5 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 6 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 15
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 15 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 16
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 16 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 25
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 25 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 26
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 26 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:153:
LysTrpLysAlaAlaAlaArgPheLeuLysLysTrpLysAlaAlaAla
151015
ArgPheLeuLysLysTrpLysAlaAlaAlaArgPheLeuLys
202530
(2) INFORMATION FOR SEQ ID NO:154:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.158"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 10 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 11
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 11 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:154:
IleLysIleSerGlyLysTrpLysAlaAlaAlaArgPheLeuLysLys
151015
SerLysValGlyTrpLeuIleGlnLeuPheHisLysLys
2025
(2) INFORMATION FOR SEQ ID NO:155:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.159"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 2 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 6 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:155:
LysAlaLysAlaGlnAlaArgPheLeuLysLysSerLysValGlyTrp
151015
LeuIleGlnLeuTrpHisLysLys
20
(2) INFORMATION FOR SEQ ID NO:156:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.160"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 2 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 6 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 12
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 12 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 16
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 16 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:156:
LysAlaLysAlaGlnAlaArgPheLeuLysLysAlaLysAlaGlnAla
151015
ArgPheLeuLys
20
(2) INFORMATION FOR SEQ ID NO:157:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.161"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:157:
LysSerLysValLysAlaLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:158:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.162"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:158:
LysTrpLysAlaGlnTrpArgPheLeuLysLysSerLysValGlyTrp
151015
LeuIleGlnLeuPheHisLysLys
20
(2) INFORMATION FOR SEQ ID NO:159:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.163"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:159:
LysTrpLysAlaGlnTrpArgPheLeuLysLysTrpLysAlaGlnTrp
151015
ArgPheLeuLys
20
(2) INFORMATION FOR SEQ ID NO:160:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.164"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 5 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 15
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 15 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:160:
LysTrpLysAlaAlaLysArgPheLeuLysLysTrpLysAlaAlaLys
151015
ArgPheLeuLys
20
(2) INFORMATION FOR SEQ ID NO:161:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.165"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 2 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 12
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 12 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:161:
LysAlaLysAlaGlnPheArgPheLeuLysLysAlaLysAlaGlnPhe
151015
ArgPheLeuLys
20
(2) INFORMATION FOR SEQ ID NO:162:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.166"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:162:
LysSerLysValGlyValLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:163:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.167"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:163:
LysTrpLysAlaGlnLysArgPhe
15
(2) INFORMATION FOR SEQ ID NO:164:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.168"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:164:
CysLysTrpLysAlaGlnLysArgPheLeuLysMetSerCys
1510
(2) INFORMATION FOR SEQ ID NO:165:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.169"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:165:
CysLysTrpLysAlaGlnLysArgPheCys
1510
(2) INFORMATION FOR SEQ ID NO:166:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.221"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 13
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 13 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:166:
IleLysIleSerGlyLysTrpLysAlaGlnLysArgAlaLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:167:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.222"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 6 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 14
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 14 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:167:
LysSerLysValGlyAlaLeuIleGlnLeuPheHisLysAla
1510
(2) INFORMATION FOR SEQ ID NO:168:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.223"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 6 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 10 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:168:
LysSerLysValGlyAlaLeuIleGlnAlaPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:169:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.224"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 6 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 9
(C) OTHER INFORMATION: /label=Substituted-Phe
/note="Position 9 is
para-amino- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:169:
LysSerLysValGlyAlaLeuIlePheLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:170:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.225"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 6 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(C) OTHER INFORMATION: /label=Substituted-Phe
/note="Position 5 is
para-amino- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:170:
LysSerLysValPheAlaLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:171:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.226"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 6 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:171:
LysSerLysValGlyAlaLeuIleGlnLeuTrpHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:172:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.227"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 10 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 14
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 14 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:172:
LysSerLysValGlyTrpLeuIleGlnAlaPheHisLysAla
1510
(2) INFORMATION FOR SEQ ID NO:173:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.228"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 9
(C) OTHER INFORMATION: /label=Substituted-Phe
/note="Position 9 is
para-amino- substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 14
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 14 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:173:
LysSerLysValGlyTrpLeuIlePheLeuPheHisLysAla
1510
(2) INFORMATION FOR SEQ ID NO:174:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.229"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 5 is
para-amino- substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 14
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 14 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:174:
LysSerLysValPheTrpLeuIleGlnLeuPheHisLysAla
1510
(2) INFORMATION FOR SEQ ID NO:175:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.230"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 14
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 14 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:175:
LysSerLysValGlyTrpLeuIleGlnLeuTrpHisLysAla
1510
(2) INFORMATION FOR SEQ ID NO:176:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.231"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 10 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 12
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 12 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:176:
LysSerLysValGlyTrpLeuIleGlnAlaPheAlaLysLys
1510
(2) INFORMATION FOR SEQ ID NO:177:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.232"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 9
(C) OTHER INFORMATION: /label=Substituted-Phe
/note="Position 9 is
para-amino- substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 12
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 12 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:177:
LysSerLysValGlyTrpLeuIlePheLeuPheAlaLysLys
1510
(2) INFORMATION FOR SEQ ID NO:178:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.233"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(C) OTHER INFORMATION: /label=Substituted-Phe
/note="Position 5 is
para-amino- substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 12
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 12 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:178:
LysSerLysValPheTrpLeuIleGlnLeuPheAlaLysLys
1510
(2) INFORMATION FOR SEQ ID NO:179:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.234"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 12
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 12 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:179:
LysSerLysValGlyTrpLeuIleGlnLeuTrpAlaLysLys
1510
(2) INFORMATION FOR SEQ ID NO:180:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.235"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 9
(C) OTHER INFORMATION: /label=Substituted-Phe
/note="Position 9 is
para-amino- substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 10 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:180:
LysSerLysValGlyTrpLeuIlePheAlaPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:181:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.236"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(C) OTHER INFORMATION: /label=Substituted-Phe
/note="Position 5 is
para-amino- substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 10 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:181:
LysSerLysValPheTrpLeuIleGlnAlaPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:182:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.237"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 10 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:182:
LysSerLysValGlyTrpLeuIleGlnAlaTrpHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:183:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.238"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(C) OTHER INFORMATION: /label=Substituted-Phe
/note="Position 5 is
para-amino- substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 9
(C) OTHER INFORMATION: /label=Substituted-Phe
/note="Position 9 is
para-amino- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:183:
LysSerLysValPheTrpLeuIlePheLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:184:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.239"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 9
(C) OTHER INFORMATION: /label=Substituted-Phe
/note="Position 9 is
para-amino- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:184:
LysSerLysValGlyTrpLeuIlePheLeuTrpHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:185:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.240"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(C) OTHER INFORMATION: /label=Substituted-Phe
/note="Position 5 is
para-amino- substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:185:
LysSerLysValPheTrpLeuIleGlnLeuTrpHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:186:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.247"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 2 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 6 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:186:
LysAlaLysAlaGlnAlaArgPheLeuLysLysSerLysValGlyTrp
151015
LeuIleLeuLeuPheHisLysLys
20
(2) INFORMATION FOR SEQ ID NO:187:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.245"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:187:
LysTrpLysAlaGlnPheArgPheLeuLysLysSerLysValGlyTrp
151015
LeuIleGlnLeuTrpHisLysLys
20
(2) INFORMATION FOR SEQ ID NO:188:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.246"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 16
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 16 is
D-beta-2- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:188:
LysTrpLysAlaGlnPheArgPheLeuLysLysSerLysValGlyAla
151015
LeuIleGlnLeuPheHisLysLys
20
(2) INFORMATION FOR SEQ ID NO:189:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.248"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 2 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 6 is
beta-1- naphthyl-substituted."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 16
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 16 is
D-beta-2- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:189:
LysAlaLysAlaGlnAlaArgPheLeuLysLysSerLysValGlyAla
151015
LeuIleGlnLeuPheHisLysLys
20
(2) INFORMATION FOR SEQ ID NO:190:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.242"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 6 is
D-beta-2- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:190:
LysSerLysValGlyAlaLeuIleLeuLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:191:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.272"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:191:
LysSerLysValGlyTrpLeuIleLeuLeuPheHisLysLysLysSer
151015
LysValGlyTrpLeuIleLeuLeuPheHisLysLys
2025
(2) INFORMATION FOR SEQ ID NO:192:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.275"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:192:
LysSerLysValGlyTrpLeuIlePheLeuPheHisLysLysLysSer
151015
LysValGlyTrpLeuIlePheLeuPheHisLysLys
2025
(2) INFORMATION FOR SEQ ID NO:193:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.270"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:193:
LysSerLysValGlyTrpLeuIleLeuLeuPheHisLysLysLysSer
151015
LysValGlyTrpLeuIleGlnLeuPheHisLysLys
2025
(2) INFORMATION FOR SEQ ID NO:194:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.271"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:194:
LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLysLysSer
151015
LysValGlyTrpLeuIleLeuLeuPheHisLysLys
2025
(2) INFORMATION FOR SEQ ID NO:195:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.273"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:195:
LysSerLysValGlyTrpLeuIlePheLeuPheHisLysLysLysSer
151015
LysValGlyTrpLeuIleGlnLeuPheHisLysLys
2025
(2) INFORMATION FOR SEQ ID NO:196:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.274"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:196:
LysSerLysValGlyTrpLeuIleGlnLeuPheHisLysLysLysSer
151015
LysValGlyTrpLeuIlePheLeuPheHisLysLys
2025
(2) INFORMATION FOR SEQ ID NO:197:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.276"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:197:
LysTrpLysAlaGlnPheArgPheLeuLysLysSerLysValGlyTrp
151015
LeuIlePheLeuPheHisLysLys
20
(2) INFORMATION FOR SEQ ID NO:198:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.241"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:198:
LysSerLysValGlyTrpLeuIleLeuLeuTrpHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:199:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.243"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 6 is
D-beta-2- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:199:
LysSerLysValGlyAlaLeuIleGlnLeuTrpHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:200:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.244"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="Position 6 is
D-beta-2- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:200:
LysSerLysValGlyAlaLeuIleLeuLeuTrpHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:201:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.249"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:201:
LysSerLysValGlyGlyLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:202:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.250"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:202:
LysSerLysValGlyLeuLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:203:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.251"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:203:
LysSerLysValGlyIleLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:204:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.252"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label=D- Ala
/note="The amino acid at position 6 is
D-alanine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:204:
LysSerLysValGlyAlaLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:205:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.253"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label=D- Val
/note="The amino acid at position 6 is
D-valine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:205:
LysSerLysValGlyValLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:206:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.254"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label=beta-Ala
/note="The amino acid at position 6 is
beta- alanine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:206:
LysSerLysValGlyAlaLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:207:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.255"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label=delta-aba
/note="The amino acid at position 6 is
delta- aminobutyric acid"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:207:
LysSerLysValGlyXaaLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:208:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.256"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label=gaba
/note="The amino acid at position 6 is
gamma- aminobutyric acid"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:208:
LysSerLysValGlyXaaLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:209:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.257"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label=d- methyl-A
/note="The amino acid at position 6 is
delta-Methyl- alanine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:209:
LysSerLysValGlyAlaLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:210:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.258"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label=t- butyl-G
/note="The amino acid at position 6 is
tert-butyl- glycine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:210:
LysSerLysValGlyGlyLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:211:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.259"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label=N- methyl-G
/note="The amino acid at position 6 is
N-Methyl- glycine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:211:
LysSerLysValGlyGlyLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:212:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.260"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label=N- methyl-V
/note="The amino acid at position 6 is
N-Methyl- valine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:212:
LysSerLysValGlyValLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:213:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.261"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /label=N- methyl-L
/note="The amino acid at position 6 is
N-Methyl- leucine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:213:
LysSerLysValGlyLeuLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:214:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.262"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:214:
LysSerLysValGlyTrpLeuIleAsnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:215:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.263"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:215:
LysSerLysValGlyTrpLeuIleGluLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:216:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.264"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:216:
LysSerLysValGlyTrpLeuIleAspLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:217:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.265"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:217:
LysSerLysValGlyTrpLeuIleLysLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:218:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.266"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:218:
LysSerLysValLysValLeuIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:219:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.267"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:219:
LysSerLysValLysTrpAlaIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:220:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.268"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:220:
LysSerLysValGlyValAlaIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:221:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.269"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:221:
LysSerLysValLysValAlaIleGlnLeuPheHisLysLys
1510
(2) INFORMATION FOR SEQ ID NO:222:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.277"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 2
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 2 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:222:
LysAlaLysAlaGlnPheArgPheLeuLysLysSerLysValGlyTrp
151015
LeuIleLeuLeuPheHisLysLys
20
(2) INFORMATION FOR SEQ ID NO:223:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.278"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:223:
IleLysIleSerGlyLysTrpLysAlaAlaTrpArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:224:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.279"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 10 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:224:
IleLysIleSerGlyLysTrpLysAlaAlaPheArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:225:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.280"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:225:
IleLysIleSerGlyLysTrpLysAlaAlaPheArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:226:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.281"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 10
(C) OTHER INFORMATION: /label=Substituted-Ala
/note="The alanine at position 10 is
beta-1- naphthyl-substituted."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:226:
IleLysIleSerGlyLysTrpLysAlaAlaAlaArgPheLeuLys
151015
(2) INFORMATION FOR SEQ ID NO:227:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "XMP.170"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:227:
LysTrpLysAlaGlnLysArgPheLeuLysMetSer
1510
(2) INFORMATION FOR SEQ ID NO:228:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "LBP-10"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:228:
GAGATCGATAGTTTCGCCGAC21
(2) INFORMATION FOR SEQ ID NO:229:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "LBP-11"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:229:
ACTATCGATCTCTGTTGTAA20
(2) INFORMATION FOR SEQ ID NO:230:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "LBP- BSM"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:230:
GAATGCAGCCAACCCCGGCTTGGTCGCCA29
(2) INFORMATION FOR SEQ ID NO:231:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "LBP-8"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:231:
CTGGCTAACCGTGGGACG18
(2) INFORMATION FOR SEQ ID NO:232:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "BPI-63"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:232:
AAAATCGATTCTGTGGCTGG20
(2) INFORMATION FOR SEQ ID NO:233:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "BPI-64"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:233:
AGAATCGATTTTGGTCATTA20
(2) INFORMATION FOR SEQ ID NO:234:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "BPI-40"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:234:
TGATCTGAAGCTGGGCAG18
(2) INFORMATION FOR SEQ ID NO:235:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "BPI-7"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:235:
GAACTTGGTTGTCAGTCG18
(2) INFORMATION FOR SEQ ID NO:236:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "LBP Peptide 1-2"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:236:
GlyGlnGlyLeuSerLeuSerIleSerAspSerSerIleArgValGln
151015
GlyArgTrpLysValArgLysSerPhePheLys
2025
(2) INFORMATION FOR SEQ ID NO:237:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: misc.sub.-- feature
(D) OTHER INFORMATION: "LBP Peptide 2-1"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:237:
AspValGluValAspMetSerGlyAspLeuGlyTrpLeuLeuAsnLeu
151015
PheHisAsnGlnIleGlu
20
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